Methods and compounds to inhibit enveloped virus release

ABSTRACT

A compound having an antiviral activity for inhibiting release of an enveloped virus from a cell is disclosed, including methods of inhibiting release of an enveloped virus from a cell. The antiviral activity of the compound includes inhibiting formation of an associative complex or disrupting formation of an associative complex. The associative complex comprises an L-domain motif of the enveloped virus and at least one cellular polypeptide, or fragment thereof, capable of binding the L-domain motif of the enveloped virus.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims benefit of priority to U.S. provisionalapplication No. 61/745,336 filed on Dec. 21, 2012, which is incorporatedby reference in its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

Not applicable.

SEQUENCE LISTING

The instant application contains a Sequence Listing that has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII copy, created in final form onDec. 20, 2013, is named NWNO1-012-US ST25.txt, and is 68,266 bytes insize.

FIELD OF THE INVENTION

This invention relates to methods for identifying compounds that inhibitinteractions with the Nedd4 family of ubiquitin ligases and Tsg101,including inhibitors of virus budding.

BACKGROUND OF THE INVENTION

There is considerable interest developing antiviral reagents to combatviral infections. The two most prevalent antiviral strategies focus oncreating immunity to viral infection by use of vaccines or byinterfering with a necessary virus-specific process essential to virusmaintenance, replication and propagation in the host.

Vaccines have been successfully developed for many viruses to combatviral infections. So-called live vaccines containing attenuatedversion(s) of the target virus provide a convenient means of conferringimmunity as typically only one inoculation is required. The drawbacks tomost live virus vaccines lie in their limited shelf life, therequirement for maintaining appropriate storage conditions to preservethe vaccine reagent, and the possibility of revertance to high virulencedue to their active replication. These drawbacks can be avoided by usingso-called inactivated virus vaccines containing a completely inert virusparticle or a sub-viral component like a protein. The drawback toinactive viral vaccines is that multiple inoculations are required toconfer full immunity. Furthermore, vaccines have an attendant risk thatadverse reactions might arise in certain populations followingimmunization (for example, autoimmunity responses associated withGuillain-Barré syndrome (GBS)).

Antiviral compounds that specifically target a viral replication processhave also proven effective for treating some virus infections. Examplesof such reagents include small molecule inhibitors selective for a givenviral protein, such as a viral replicase (for example, the nucleosideanalog 3′-azidothymidine for inhibiting the HIV-1 reverse transcriptase)or a viral protease (for example, Darunavir for inhibiting HIV-1protease). Owing to their small molecular size and chemical composition,antiviral compounds can be formulated as pharmaceutical compositionshaving significant shelf life and can typically retain their potencyover a larger temperature range during storage than many vaccines.However, HIV-1 and other virus can mutate to escape the effectiveness ofthe antiviral drugs when such drugs are targeted against virus-specificproteins. In particular, HIV-specific drugs have side-effects that causepatients to interrupt therapy that can lead to drug-resistant viralstrains.

Generally, antiviral compounds are typically used in combinations formaximum efficacy and durability. Though most aspects of the viralreplication process are susceptible to targeting and inhibition, theprimary focus of antiviral inhibitor drug development is on early stageprocesses of viral replication, when the copy number of viral protein ornucleic acid targets is relatively low.

Late stage replication events include those associated with virusparticle assembly and release from the host cell. These viral processesare more difficult targets to develop antiviral reagents. This is due inpart to the vastly larger number of virus particles that result fromactive viral replication.

Enveloped virus particles adopt an outer membrane structure composed ofthe host cell membrane in its final virus form. Examples of envelopedviruses include retroviruses (for example, human immunodeficiency virus,type 1), rhabdoviruses (for example, rabies virus), and herpes viruses(for example, herpes simplex virus, type 1). For enveloped viruses, thefinal stages of virus replication include envelope maturation, buddingand release.

No antiviral therapeutic reagents have been developed that target theprocesses of enveloped virus budding and release. This is due in largepart to the inability to target virus-specific proteins, owing to thelarge number of viral proteins present during late phase infection. Butmore importantly, the host cell-virus interactions responsible forenveloped virus particle maturation, budding and release are only poorlyunderstood.

BRIEF SUMMARY OF THE INVENTION

In a first aspect, a compound having an antiviral activity forinhibiting release of an enveloped virus from a cell is disclosed. Inone aspect, the compound has an antiviral activity that includesinhibiting formation of an associative complex or disrupting formationof an associative complex. The associative complex comprises an L-domainmotif of the enveloped virus and at least one cellular polypeptide, orfragment thereof, capable of binding the L-domain motif of the envelopedvirus.

In a second aspect, a method of inhibiting release of an enveloped virusfrom a cell is disclosed. The method includes the step of contacting thecell with a compound having an antiviral activity. The antiviralactivity includes inhibiting formation of an associative complex ordisrupting formation of an associative complex. The associative complexcomprises an L-domain motif of the enveloped virus and at least onecellular polypeptide, or fragment thereof, capable of binding theL-domain motif of the enveloped virus.

In a third aspect, a pharmaceutical composition comprising a compoundhaving an antiviral activity for inhibiting release of an envelopedvirus from a cell and optionally a pharmaceutically acceptable carrieris disclosed.

BRIEF DESCRIPTION OF THE DRAWINGS

The application file contains at least one drawing executed in color.Copies of this patent or patent application publication with colordrawing(s) will be provided by the Office upon request and payment ofthe necessary fee.

FIG. 1 depicts parallel pathways used by ASLV and HIV-1 Gag to bud fromcells.

FIG. 2 depicts a first embodiment to screen in vitro for the ability ofa test compound (104) to inhibit an associative complex (103) formedbetween a fluorescently-labeled viral peptide probe (101) containing aviral L-domain motif and a cellular ESCRT complex protein (102). Thefluorescence of 101 in 103 is denoted as F₁, while the fluorescence of101 free is denoted as F₂. Note that compound 104 need not bind to thesame site as probe 101 on protein 102 or that probe 101 need becompletely dissociated from 102 to have F₂ arise.

FIG. 3 depicts a second embodiment to screen in vitro for the ability ofa test compound (204) to bind to a cellular ESCRT complex protein (202)that is immobilized to substrate (201) via a linking group (203). Theassay measures changes in the index of refraction upon a binding event(205) by comparing the wavelength of the reflected light (RL₁) in theabsence of 204 to the wavelength of the reflected light (RL₂) in thepresence of 204.

FIG. 4 depicts a third embodiment to screen in vivo for the ability of atest compound (304) to inhibit a reconstituted EGFP two-hybrid complex(303) formed between a partial N-terminal fusion EGFP polypeptide(denoted by small ball) that includes a viral peptide (301) containing aviral L-domain motif and a complementary partial C-terminal EGFP fusionpolypeptide that includes a cellular ESCRT complex protein or a viralL-domain binding domain derived therefrom (302). The reconstituted EGFPcomplex 303 produces the fluorescence (Fl) analogous to native EGFP when301 and 302 interact in vivo. If test compound 304 interferes withformation of 403, the cells produce less fluorescence.

FIG. 5A depicts EPIC label-free binding assay data with Celastrol andWWP2 (SEQ ID NO:24).

FIG. 5B depicts EPIC label-free binding assay data with Oxytetracyclineand WWP2 (SEQ ID NO:24).

FIG. 5C depicts EPIC label-free binding assay data with Benserazide HCland WWP2 (SEQ ID NO:24).

FIG. 6A depicts an exemplary assay that shows the effect of smallmolecule inhibitors targeting Nedd4 interaction with PPPY-containingviral Gag motif L-domain on the release of ASLV virus like particles(VLPs) from 293 cells, as adjudged by Western blot assay using ananti-ASLV Gag antibody to detect ASLV Gag protein that remainsassociated with the cells (cell lysates) or released from cells (VLPs)when 293 cells that express ASLV Gag protein are contacted with noinhibitor or with inhibitors (K21 [Benserazide Hydrochloride] and N20[Oxytetracycline]) at the indicated concentrations in the media. TheASLV Gag protein denoted as Δp2b contains a deletion of the L-domainmotif. The budding defect can be seen by comparing release of VLPs fromuntreated cells (lane 2) to K21-contacted cells (lanes 3-5) andN20-contacted cells (lanes 6-8). Lane 1 is loading control for untreatedcells that do not express ASLV Gag protein. Lane 9 is a control foruntreated cells that express ASLV Gag with an L-domain deletion (Δp2b).These inhibitor concentrations are not cell toxic.

FIG. 6B depicts percentage of absorbance of control (0 μM) as a functionof concentration of the inhibitor K21 [Benserazide Hydrochloride] in themedia.

FIG. 6C depicts VLP release (LOG(PFU/ml) as a function of time for theindicated concentrations of the inhibitor K21 [BenserazideHydrochloride] in the media.

FIG. 6D depicts VLP release (LOG(PFU/ml) as a function of time for theindicated concentrations of the inhibitor K21 [BenserazideHydrochloride] in the media.

FIG. 7 depicts an fluorescence-based thermal shift (FTS) assay to detecta thermal shift in protein folding of TSG101 (SEQ ID NO:33) due to thepresence of inhibitor compound N16. In this case, the inhibitor compoundinduced thermal instability, resulting in a lower T_(m) for proteinunfolding.

FIG. 8A depicts results of infectious HSV-1 virion production from HSV-1infected VERO cells alone or HSV-1 infected VERO cells contacted withPTAP-motif inhibitor compounds, F15 (Esomeprazole potassium) and N16, atthe indicated concentrations. The titers of infectious HSV-1 virusparticles was determined from plaque assays with naïve (uninfected) VEROcell cultures infected with virions harvested from either the culturedcell media containing the VERO cells contacted with no inhibitorcompound or with one of compounds F15 or N16 (“supernatant”), or fromboth the cultured cell media and the VERO cells contacted no inhibitorcompound or with one of compounds F15 or N16 (“total”). The indicatedconcentrations of the PTAP-motif inhibitor compounds, F15 and N16, arenot toxic to the contacted VERO cells.

FIG. 8B depicts results of MTS-based assays for evaluating cytotoxicityof cells contacted with the PTAP-motif inhibitor compounds, F15 and N16,where Absorbance at 490 nm (indicative of cell viability) is plotted asa function of inhibitor concentration present in cell culture medium incontact with the cells.

DETAILED DESCRIPTION OF THE INVENTION

Disclosed herein are methods and materials to inhibit the interaction ofthe above-mentioned cellular proteins or fragments thereof with Ldomain-containing peptides of enveloped viruses. Applicants made theseminal discovery that enveloped viruses use cellular pathways formediating virus budding and that inhibiting these pathways results insignificantly decreased rates of enveloped virus release from cellsurfaces. The methods disclosed herein provide a robust, high-throughputapproach to identify lead compounds having potent inhibitory effects onenveloped virus protein interactions with the components of thesepathways and, thereby, virus particle release.

Referring to FIG. 1, enveloped viruses such as avian sarcoma andleukosis virus (ASLV) and human immunodeficiency virus, type 1 (HIV-1)include late assembly domains (“L-domains”) encoded within their Gagprotein sequence that interact with cellular components of the endosomalsorting complex required for transport (“ESCRT”) machinery for virusbudding and release from cells. The L-domains have been identified in avariety of enveloped viruses and families of enveloped viruses.Applicants have identified a consensus subset of L-domain motifs thatinteract with the critical ESCRT-dependent processes that envelopedviruses use to bud from cell membranes (see Table I, underlined, boldsequences).

TABLE I L-domains found in enveloped virus proteins. Amino acid sequenceSEQ Virus Pro- containing the L- ID species Virus¹ tein domain² NO:Arenavirus LFV Z AA PTAP PTGAADSI PPPY SP  1 LCMV Z TAPSS PPPY EE  2Filovirus EboV VP40 MRRVIL PTAPPEY MEAI  3 MarV VP40 NTYMQYLN PPPY ADHS 4 Hepadnavirus HBV Core PPAY  5 Herpesvirus HSV-1 E PPTY  6 HSV-2 UL56PPPY  7 CMV UL32 PTAP  8 Paramyxovirus SV5 M QSIKA FPIV INSDG  9 MuV MRLNA FPIV MGQ 10 Retrovirus ASLV p2B ATASA PPPPY VGSG LYP S L 11 (Gag)HIV-1 p6 PE PTAP PFLQSRPE PTAP PEES 12 (Gag) HTLV- MA DPQI PPPY VE PTAP13 I (Gag) EIAV p9 QNL YPDL SEIK 14 (Gag) Rhabdovirus VSV M LGIA PPPYEEDTSMEYA PSAP 15 RV M DDLWL PPPEY VPLKEL 16 ¹Virus names correspondingto the abbreviations presented are as follows: LFV, Lassa fever virus;LCMV, lymphocytic choriomeningitis virus; EboV, Ebola virus; MarV,Marberg virus; HBV, hepatitis B virus; HSV-1, Herpes simplex virus, type1; HSV-2, Herpes simplex virus, type 2; CMV, cytomegalovirus; SV5,Simian virus, type 5; MuV, Mumps virus; ASLV, avian sarcoma leucosisvirus; HIV-1, human immunodeficiency virus, type 1; HTLV-I, humanT-lymphotrophic virus, type 1; EIAV, equine infectious anemia virus;VSV, vesicular stomatitis virus; and RV, rabies virus. ²Underlined,bolded sequences indicate the consensus sequences within the L-domains.

One of these L-domain motifs, termed the PTAP motif (for example, fromHIV-1), interacts with the TSG101 protein that becomes recruited as partof the ESCRT complexes. Another of these L-domain motifs, termed PPPYmotif (also referred to as the “PY motif” or the “PY L-domain motif;”for example, from ASLV), interacts with the Nedd4 family of proteinsthat is also recruited by ESCRT-associated proteins. While it is oftenthe case that certain viruses have a viral protein might encode bothtypes of L-domains, typically only one predominates in the viral buddingprocess through interactions with ESCRT machinery. The Applicants havedevised novel, robust screening methods to identify compounds thatinterfere with the interaction between viral L-domains that include thePPPY motif or PTAP motif and ESCRT component, TSG101 or ESCRT-linkedcomponent, Nedd4 family proteins. These screening methods enable one torapidly identify compounds that inhibit the interactions of both Nedd4and TSG101 with the viral L-domain motifs, thereby providing ahigh-throughput strategy to obtain candidate lead compounds havingutility as novel antiviral agents for inhibiting virus budding andrelease from infected cells.

Some candidate lead compounds can display potency at inhibiting onlyNedd4- or TSG101-mediated ESCRT pathways, thereby offering specificantiviral activity for one type of virus or virus family. Yet othercandidate lead compounds can display potency at inhibiting both Nedd4-or TSG101-mediated ESCRT pathways, thereby offering broad-spectrumantiviral activity to a plurality of diverse enveloped virus families.Thus, the screening methods disclosed herein contemplate identificationof compounds having either narrow- or broad-spectrum antiviral effects.

One preferred embodiment of such a screening method is depicted in FIG.2. Briefly, the assay is based upon changes in the fluorescencepolarization of a fluorescently-labeled viral peptide probe 101 thatincludes an L-domain motif in an associative complex 103 with selectedcellular ESCRT-associated protein 102 (for example, TSG101 (SEQ ID NOs:32 or 33) or a Nedd4 family polypeptide (for example, WWP1 (SEQ IDNO:29), WWP2 (SEQ ID NO:24), Nedd4 L, among others), or a fragmentthereof) as a function of the presence or absence of test compound 104.Because the associative complex 103 restricts motion of thefluorescently labeled viral peptide probe 101, the latter displayscharacteristic fluorescent polarization properties (denoted by F₁ ofFIG. 2). Upon introduction of compound 104 into solutions containing theassociative complex 103, an interaction between compound 104 and theassociative complex 103 that alters or releases thefluorescently-labeled viral peptide probe 101 will result in a change influorescence polarization of the solution (denoted F₂ of FIG. 2.)

Permutations and variations of the embodiment of the screening assay(FIG. 2) are recognized to one skilled in the art based upon thisdisclosure and fall within the scope of the invention. For example, theselection of the fluorescent label and its attachment within probe 101can be varied, provided that associative complex 103 can form having adiscernible fluorescence polarization signal. For example labels can beintroduced at the amino terminus, the carboxy terminus or at suitablelocations within the peptide probe using coupling chemistries that arewell known in the art. In particular, the type of fluorescent labelselected will depend upon a variety of factors, such as whether thespectral properties of the label can be discerned under assay conditionssuch as the presence of fluorescent species derived from assayscomponents like the viral peptide sequences that form probe 101, theselected cellular ESCRT protein 102, and the test compound 104.

Likewise, the preferred choices of the viral peptide sequences forinclusion in probe 101 are routine in nature, as are the preferredchoices of whether a single copy or a plurality of copies of said viralsequences are to be included in probe 101. For example, threetandemly-linked copies of the PPPY motif from the Δp2b Gag protein ofALSV (for example, SEQ ID NO:17) provided a K_(d) of 0.20 μM for theWWP2 polypeptide (SEQ ID NO:24), as compared to the correspondingmonomer that had a K_(d) of 18 μM. Though any specific viral peptidesequence can be included in probe 101, preferred embodiments includeL-domain motifs, including those of Table I (SEQ ID NOS: 1-16) andESCRT-linked protein (for example, Nedd4 family polypeptides (forexample, WWP1 (SEQ ID NO:29), WWP2 (SEQ ID NO:24), Nedd4 L, amongothers) and TSG101 polypeptide, or a fragment thereof), binding variantsthereof, as well as others known in the art.

Furthermore, additional representative viral L-domain motifs can beidentified from a variety of viruses belonging to different envelopedvirus families using standard biochemical and molecular techniques.These L-domain motifs can be screened using modifications of the assaypresented in FIG. 2. Representative viruses for this purpose includeLassa fever virus; lymphocytic choriomeningitis virus; Ebola virus;Marberg virus; West Nile Virus; hepatitis B virus; Herpes simplex virus,type 1; Herpes simplex virus, type 2; cytomegalovirus; Simian virus,type 5; Mumps virus; avian sarcoma leucosis virus; humanimmunodeficiency virus, type 1; human T-lymphotrophic virus, type 1;equine infectious anemia virus; vesicular stomatitis virus; and rabiesvirus, among others.

Likewise, the choice of cellular ESCRT component polypeptides of Nedd4family members (for example, WWP1 (SEQ ID NO:29), WWP2 (SEQ ID NO:24),Nedd4 L, among others) and TSG101 depends upon the solubility attributesof the selected protein. For example, while the recombinant Nedd4polypeptides are soluble under assay conditions, the full-length TSG101was not. In the case of conducting this screening assay with TSG101, asoluble recombinant polypeptide fragment derived from the full-lengthprotein having the binding domain for the viral L-domain motifs wasprepared and used for screening assays (See SEQ ID NO:33, Table VI). Oneparticularly sensitive assay for detecting candidate inhibitorinteractions with TSG101 (SEQ ID NO:33) is based upon the ability of thecandidate to alter the thermal denaturation profile of protein foldingfor a soluble fragment of TSG101 peptide (SEQ ID NO:33), as assessed byfluorescence methods. These assays and other aspects are described indetail in the Examples.

A high-throughput assay using the screening method of FIG. 2 was devisedto screen a library of 70,000 compounds for the ability to disruptcomplexes formed between Nedd4 WWP2 (SEQ ID NO:24) and the ASLV L-domainmotif containing PPPY (for example, SEQ ID NO: 17). The results of thisinitial screen (for example with FITC-labeled probe 101 (SEQ ID NO:18))yielded about 700 candidate compounds having the desired molecularinhibitory properties. Owing to the possibility of false-positivesarising in these assays, the initial collection candidate compounds arerescreened in a secondary assay fitted with viral probe 101 containing adifferent fluorescent label (for example, with TAMRA-labeled probe 101(SEQ ID NO:19)). The results of the secondary screen with the identifiedcollection of 700 compounds provided a further reduction of viablecandidates to 130 compounds having properties for inhibiting virusrelease from cells.

Another preferred embodiment of an in vitro screening method is depictedin FIG. 3. In this method, the ability of the candidate compound 204 todirectly interact with the cellular ESCRT-linked component protein (forexample, Nedd4 family of polypeptides [for example, WWP1 (SEQ ID NO:29),WWP2 (SEQ ID NO:24), Nedd4 L, among others]) or TGS101 is evaluated.According to this embodiment, a cellular ESCRT component protein 202 ispreferably immobilized to a substrate 201 (for example, a microtiterplate) via a linking group 203 (for example, an amine group). Thesubstrate preferably includes an optional reference area that provides acontrol to prevent non-specific interactions with non-mobilized ESCRTcomplex protein 202 with substrate 201. The substrate 201 is washed andirradiated with broadband light so that a baseline refractive index ismeasured. Test compound 204 is added to the system to permit its bindingto the immobilized ESCRT component protein 202 to form a resultantassociative complex 205. The substrate 201 is irradiated with broadbandlight again to detect a change in the wavelength of the reflected light.

Commercial instruments are available to enable high-throughput screeningof test compounds 204 for their ability to bind ESCRT component protein202 (for example, TSG101 (SEQ ID NOs: 32 or 33) or a Nedd4 familypolypeptide (for example, WWP1 (SEQ ID NO:29), WWP2 (SEQ ID NO:24),Nedd4 L, among others)) for example, the Epic® technology; PerkinElmer,Inc. (Watham, Mass.)). As will be apparent to one skilled in the art,either the ESCRT component protein 202 or the test compound 204 may beimmobilized to substrate 201. However, it is preferable to immobilizeESCRT component protein 202 to provide a high-throughput platform forscreening a plurality of test compounds 204 in parallel for theirability to form associative complex 205. These assays and other aspectsare described in detail in the Examples.

This screening method can be used in conjunction with the methodoutlined in FIG. 2 to further refine the collection of identifiedcompounds to identify those that directly interact with ESCRT componentpolypeptides. For example, the screening method was applied to 30candidates from the collection of 130 compounds identified previously toprovide seven compounds having the ability to bind directly to Nedd4family polypeptides in the micromolar range of inhibitor compoundconcentrations. As further described below, two of these compounds werefound to be non-toxic to cultured cells and were capable of blockingPPPPY-dependent virus-like particle (VLP) budding and release fromcells.

The advantages of performing the screening method of FIG. 3 (relative,for example, to the embodiment illustrated in FIG. 2) is that one canmeasure direct binding interactions between test compounds 204 and ESCRTcomponent proteins 202 in a label-free assay. In addition to being ableto detect direct biomolecular interactions between test compounds 204and ESCRT component proteins 202, one can rapidly perform thermodynamicmeasurements of the binding interaction (for example, K_(d)determinations) resulting in formation of associative complex 205. Thislatter advantage can provide an estimate of whether certain compounds(204) display viable binding properties supportive of continuing on withbiological testing of the compounds in viral inhibition studies.

FTS Assay for Detecting Direct Binding Interactions Between TestCompounds and ESCRT Component Proteins.

The fluorescence-based thermal shift assay is based on the observationthat a protein unfolds upon heating, exposing the hydrophobic residueswithin its tertiary structure. The unfolding temperature (T_(m)) isdetermined by the protein's primary sequence and solution environment.The FTS assay uses a fluorescent dye sensitive to a hydrophobicenvironment to probe protein stability and its modulation by smallmolecule ligands. The dye has a low fluorescence quantum yield when in apolar environment. Once in contact with the hydrophobic core normallyburied within a folded protein that has become exposed during thethermal unfolding (melting) process, the quantum yield of the dyeincreases, thus providing a reporting signal. Furthermore, a protein'sstability can be affected by ligand binding, resulting in an increase ordecrease in its melting temperature. FTS assay uses the Tm shift uponbinding of a ligand to identify hit compounds for drug discovery. SeePantoliano M W, Petrella E C, Kwasnoski J D, Lobanov V S, Myslik J, GrafE, Carver T, Asel E, Springer B A, Lane P, Salemme F R. (2001)High-density miniaturized thermal shift assays as a general strategy fordrug discovery. J Biomol Screen 6: 429-440; Lo M C, Aulabaugh A, Jin G,Cowling R, Bard J, et al. (2004) Evaluation of fluorescence-basedthermal shift assays for hit identification in drug discovery. AnalBiochem 332: 153-159, which are incorporated by reference in theirentireties.

In Vivo Screening Methods-Based Molecular Genetic Assays, Cell-Based VLPProduction Assays and Whole-Virus Replication Assays

As an alternative or complementary embodiment to the described screeningmethods in vitro, an in vivo screening method is contemplated asdepicted in FIG. 4. In this method, EGFP is genetically engineered astwo-half polypeptide gene cassettes (that is, N-terminal portion of EGFPand C-terminal portion of EGFP), which when expressed simultaneously inthe cells, fail to provide a functionally reconstituted EGFP havingfluorescence properties. The two-half EGFP polypeptide gene cassettesare engineered to include a viral L-domain motif (for examples, a PYmotif or a PTAP motif) in one of the EGPF cassettes (see FIG. 4, 301)and a cellular ESCRT component protein (for example, a Nedd4 familypolypeptide or a TSG101 polypeptide or fragment thereof (SEQ ID NOs: 32or 33)) in the other EGFP cassette (see FIG. 4, 302). When cells areco-transfected with expression vectors containing half-EGFP genecassettes, the corresponding fusion proteins are expressed to enablereconstitution of EGFP functional activity via interaction between thetwo heterologous fusion partners, the viral L-domain motif and thecorresponding ESCRT complex polypeptide in the form of an associativecomplex (see FIG. 4, 303). Cells (either transiently or stablyexpressing the EGFP constructs) can be evaluated using test compounds(see FIG. 4, 304) to assess whether the compounds reduce cellularfluorescence. These assays and other aspects are described in detail inthe Examples.

Candidate compounds having an inhibitory effect of fluorescence havealso been evaluated for their ability to interfere with normal cellularphysiology and growth by, for example, determining cytotoxicity profilesof the compounds as a function of dose response and incubation time withthe cells. One advantage of the in vivo assay is that it providesadditional opportunities to survey test compounds that otherwise mightnot be possible with the aforementioned biochemical assays (for example,with assays involving certain ESCRT component polypeptides havinglimited solubility in vitro). Other further advantages of in vivo assaysof this sort is that they can provide a useful model for studyingcompound transport and clearance in cells as would be important fordetermining ADME profiles (for examples, bioactivity, bioavailability,bio-inactivation, among others) at a cellular level, as well as provideadditional confirmatory evidence of the biological potency of thecompounds in a more meaningful, biological context.

For candidate lead compounds identified through one or more of theaforementioned screening methods, biological assays have beenestablished to evaluate the specific antiviral inhibitory effects thecompounds have on virus budding and release. In one assay, virus likeparticle (“VLP”) production can be evaluated as a function of testcompound dose. For example, human 293 cells can permit use of ASLV gagexpression systems to study viral protein expression and VLP productionas a function of test compound dose response. Likewise, human 293 cellscan be used to follow VLP production with HIV-1 gag expression systemsas a function of test compound dose response. Follow-up experiments wellwithin the skilled artisan's grasp include evaluating other aspects ofviral replication, as monitored by standard biochemical assays (PCR,RT-PCR, western blot methods and the like) as well as cell toxicityeffects. These assays and other aspects are described in detail in theExamples or are otherwise well understood in the art.

VLP production assays have provided evidence of candidate lead compoundsshowing antiviral inhibitory effect on virus particle release as afunction of dose, experiments then can proceed to demonstrate theantiviral effect in whole virus replication assays. Three preferredwhole virus replication assay systems include the rhabdovirus VSVreplication system, the herpes virus KSHV replication system, HSV-1replication assay in Vero cells, and HIV-1 virus replication system withassays in the physiological host, i.e., CD4+ T cells. These assays andother aspects are described in detail in the Examples.

The aforementioned in vitro and in vivo screening methods can becombined either in series or in parallel (and in any order) to identifycompounds having either narrow-spectrum activity against a few virusesor broad-spectrum antiviral activity against many different viruses. Forexample, lead compounds identified that interact with Nedd4 familymembers can be evaluated for their ability to interact with TSG101 or todisrupt TSG101—viral L-domain interactions. In this manner, differentantiviral compounds can be discerned having discrete types of inhibitoryactivity. Further, one can identify gradients of antiviral potencyacross entire classes of viruses by evaluating the dose responseprofiles in a combination of biochemical and biological assays withdifferent virus families having different viral L-domain motifs, asdescribed herein. Moreover, combinations of compounds haveTSG101-specific inhibitory activity and Nedd4 family-specific inhibitoryactivity can be tested against virus infection to determine whether thedrug combinations block virus access to the ESCRT-complex dependentpathways are blocked for enveloped virus release.

These approaches have clear utility for two simple reasons. First,L-domains encoding the aforementioned PY motifs and PTAP motifs can befound with viral proteins for single virus families. Thus, viruseshaving both types of L-domains can potentially utilize both pathwaysmediated by Nedd 4 and TSG101. Second, the L-domains used by viruses areinterchangeable. Thus, there is a need for compounds to disrupt bothinteractions between viral L-domains with the two different pathwaysmediated by Nedd 4 and TSG101, wherein virus budding and release canoccur from different cellular membranes.

The identified compound inhibitors have utility as antiviral therapeuticagents. The therapy is a post infection treatment that will slow downthe spread of virus by preventing particles from releasing from infectedcell surfaces. The accumulation of particles will enhance detection bythe immune system, which will clear the infection. The human bodyalready has an innate immunity response that targets the release ofvirus particles late in infection. Thus, the above approach hasviability because it will complement the natural immunity mechanism.

By using the in vivo screening assays described herein, severalcompounds have been identified having inhibitory effects on viralL-domain motif interaction with ESCRT component polypeptides, or in thealternative, having the capability to bind directly to the ESCRTcomponent polypeptides. These compounds are listed in Tables II-IV.

TABLE II Candidate compounds that inhibit viral budding and release.Relevant Compound Assay Identification¹ Property² NSC306711; FP Assay(FITC/TAMRA); 1 μM 813419-93-1 Epic; VLP-ASLV NSC128437 FP Assay(FITC/TAMRA); 3 μM Epic Assay CD27-G10 FP Assay (FITC/TAMRA); 1 μM EpicAssay CD23-G07 FP Assay (FITC/TAMRA); 20-30 μM    Epic Assay CD15-B10 FPAssay (FITC/TAMRA); Epic Assay ¹Assays used include the fluorescencepolarization assay (“FP Assay”) with SEQ ID NO: 17 coupled to either aFITC label (SEQ ID NO: 18) or a TAMRA label (SEQ ID NO: 19)(“FITC/TMR”); label-free, refractive index assay (“Epic Assay”).²Relevant property is [compound] to achieve 50% change in fluorescencepolarization in FP Assay.

Candidate compound NSC306711; 813419-93-1 has the following structure(I):

Candidate compound NSC128437 has the following structure (II):

Candidate compound CD27-G10 has the following structure (III):

Candidate compound CD23-G07 has the following structure (IV):

Candidate compound CD15-B10 has the following structure (V):

Additional candidate compounds were identified with the fluorescencepolarization assay and Epic label-free binding assay using WWP2 (SEQ IDNO:24) as the target. These candidate compounds and their properties arepresented in Table III. The respective binding assays using the Epiclabel-free system are depicted in FIG. 5.

TABLE III Candidate compounds that inhibit PY-motif interactions withWWP2 Kd IUPAC Name (μM) (Common Name) CAS Reg. No. Structure 18.53-Hydroxy-9β,13α- dimethyl-2-oxo-24,25,26- trinoroleana-1(10),3,5,7-tetraen-29-oic acid (Celastrol) 34157-83-0

10.6 (4S,4aR,5S,5aR,6S,12aS)-4- (dimethylamino)- 3,5,6,10,11,12a-hexahydroxy-6-methyl- 1,12-dioxo- 1,4,4a,5,5a,6,12,12a-octahydrotetracene-2- carboxamide (Oxytetracycline) 79-57-2

 7.9 (RS)-2-amino-3-hydroxy-N′- (2,3,4- trihydroxybenzyl)propanehy-drazide hydrochloride (Benserazide Hydrochloride) 322-35-0

Additional candidate compounds were identified with the fluorescencepolarization assay and Epic label-free binding assay using a solublefragment of TSG101 (SEQ ID NO:33) as the target. These candidatecompounds and their properties are presented in Table IV. Thefluorescence-based thermal shift assay is another exemplary bindingassay for identifying lead candidates (FIG. 7).

TABLE IV Candidate compounds that inhibit PTAP-motif interactions withTSG101 (SEQ ID NO: 33) IUPAC Name (Common Name) CAS Reg. No. Structure5-methoxy-2-[(R)-[(4- methoxy-3,5- dimethypyridin-2-yl)methane]sulfinyl]-1H-1,3- benzodiazole, potassium (Esomeprazolepotassium) 161796-78-7

(RS)-3-Methoxy-8-[(4- methoxy-3,5-dimethyl-pyridin-2-yl)methylsulfinyl]- 2,7,9- triazabicyclo[4.3.0]nona-2,4,8,10-tetraene (Tenatoprazole) 113712-98-4

2-Phenyl-1,2- benzoselenazol-3-one (Ebselen) 6090-34-3

(4E)-5-Methyl-2-phenyl-4- {[(2,4,6-tribromophenyl)-amino]methylene}-2,4- dihydro-3H-pyrazole-3- thione

[4-{[(4-Methylphenyl)- sulfonyl]amino}-3,6- dihydro-1,3,5-triazin-1(2H)-yl]acetic acid

These compounds were further evaluated as antiviral inhibitors of virusbudding and release using the methods disclosed herein. Cultured T cellscontacted with either Benserazide Hydrochloride (K21) or Oxytetracycline(N20) [candidate PY-binding motif inhibitors against the Nedd 4 peptidefamily members] and then subsequently infected with HIV-1 displayed asignificant reduction in HIV-1 particle release, as adjudged bydetection of HIV-1 CA protein in the cell culture media by ELISA (seeTable V).

TABLE V Activity of compounds to Inhibit HIV-1 release from T-Cells inculture HIV-1 CA protein Compound [Compound]¹ in culture media² None  0μM 3.2 ng/mL Benserazide Hydrochloride 100 μM 0.46 ng/mL Oxytetracycline  20 μM 1.9 ng/mL ¹Indicated inhibitor concentrationstested are not toxic to the contacted cells. ²Results averaged for sixexperiments, as detected by ELISA.

Vero cells were inoculated with HSV-1 (strain F) at a multiplicity ofinfection (MOI) of 0.01 for 2 hr. The cells were washed and treated with0.10 M sodium citrate buffer to inactivate viruses on the outside of thecells (for example, in the culture medium). The culture medium wasreplaced with fresh culture medium containing no inhibitor compound ordifferent concentrations (40 μM or 80 μM) of inhibitor compounds F15 orN16 (candidate PTAP motif inhibitors against the TSG101 peptide [SEQ IDNO:33]) for 48 hr. The supernatants (culture medium) and total cells(culture medium and cells) were collected for determining infectiousvirus titer. The virus titers were determined by standard plaque assayon Vero cells.

As shown in FIG. 8A, inhibitor compound F15 (Esomeprazole potassium)reduced infectious HSV-1 virion release from HSV-1 infected cells bymore than 90% as compared to infected cells not contacted with aninhibitor compound at the higher concentration tested (80 μM) Inhibitorcompound F15 (Esomeprazole potassium) also reduced the total load ofinfectious virions produced in the cells, whether released or not fromthe cells, by more than 75% relative to that observed with infectedcells not contacted with an inhibitor compound at the higherconcentration tested (80 μM).

Still referring to FIG. 8A, inhibitor compound N16 (Tenatoprazole)reduced infectious HSV-1 virion release from HSV-1 infected cells bymore than 95% as compared to infected cells not contacted with aninhibitor compound at the higher concentration tested (80 μM). Thus,inhibitor compound N16 was slightly more effective than F15 at reducinginfectious virion particle release from HSV-1 infected cells at theconcentrations tested.

Furthermore, the inhibitor compounds disrupted infectious particleassembly for virions that remained associated with cells in anunreleased state (FIG. 8A). Thus, the inhibitor compounds F15 and N16were demonstrated to inhibit HSV-1 virion release from cells.

To rule out the possibility that the reduced infectious virionproduction was attributed to the inhibitor compounds exhibiting ageneral cytotoxic effect on the host cells, cytotoxicity assays wereperformed on uninfected cells, wherein the cells were contacted withculture medium containing the inhibitor compounds in a concentrationrange from 0 μM to 100 μM. As shown in FIG. 8B, the two evaluatedinhibitor compounds, F15 and N16, did not display cytotoxic effects onthe Vero cells at the concentrations tested.

Thus, the methods disclosed herein can provide compounds havingantiviral activity for inhibiting envelope virus release from cells.Moreover, the methods provided herein can identify compounds havingantiviral activity for inhibiting formation of or disrupting anassociative complex, wherein the associative complex comprises anisolated enveloped virus L-domain motif and at least one isolatedcellular polypeptide, or fragment thereof, capable of binding theisolated virus L-domain motif. Furthermore the disclosed methods canyield compounds having binding affinity for at least one isolatedcellular polypeptide, or fragment thereof, capable of binding theisolated virus L-domain motif.

Pharmaceutical Compositions

Pharmaceutical compositions comprising a compound having an antiviralactivity for inhibiting release of an enveloped virus from a cell arecontemplated herein. Such compositions can include pharmaceuticallyacceptable carrier. Such carriers are amenable for enhancing one or moreADME characteristics, including solubility and bioavailability ofcompound inhibitors in physiologically acceptable or suitable mediasystems or biological fluids. For example, compound inhibitors havingpoor solubility can be encapsulated in nanoparticles or vesiclescomprising at least one micelle-forming lipid. Examples of such deliverysystems are disclosed in the literature, as exemplified by one or moreof the following citations, the contents of each of which are herebyincorporated by reference in their entireties: U.S. Pat. Publication No.US2002/0099164 A1 to Watterson et al.; U.S. Pat. Publication No.US2008/0008749 A1 to Pearlman et al.; and U.S. Pat. Publication No.US2013/0164379 A1 to Gartel et al.

Exemplary pharmaceutical compositions are well known in the art andfully amenable for use with the present compound inhibitors describedherein. See, for example, U.S. Pat. No. 8,202,553 to Lan et al., thecontents of which are hereby incorporated by reference in its entirety.

EXAMPLES Example 1. Materials and Methods Expression and Purification ofNedd4 Related Proteins

Construction and Purification of WWP2 Protein.

The WWP2 encoding gene was excised by EcoR1 and Xho1(NEB) cleavage fromWWP2_pCNA3.1 construct and was ligated to pET28b(+) His-tag plasmid(Novagen) by using T4 DNA ligase (NEB) in 50 mM Tris-HCL (pH 7.5), 10 mMMgCl₂, 10 mM dithiothreitol, 1 mM ATP, 25 mg/ml bovine serum albumin at16° C. overnight. E. coli. BL21 DE3 cells (Invitrogen) were transformedwith WWP2_pET28b and were expressed protein by 0.1 mM (final) IPTGinduction for 3 hrs at 25° C. His-tagged WWP2 proteins were purifiedwith His-Bind column (Novagen). Proteins were purified by manufacturer'sprotocol. Cells were lysed by sonication for 2 min at 4° C. in thepresence of 1× binding buffer (20 mM Tris-HCl, 0.5 M NaCl, 5 mMImidazole, pH 7.9) with a proteinase inhibitor cocktail-EDTA free(Roche), 0.1% NP40, and 1 mM PMSF. The cell debris and inclusion bodieswere pelleted by centrifugation at 9000 rpm for 8 min. The supernatantfraction was then passed through a 0.45 μm filter and loaded onto aNi⁺²-NTA His-Bind column at 4° C. The column was washed with Bindingbuffer (10 times bed volume of resin) followed by an equivalent amountof Wash buffer (20 mM Tris-HCl, 0.5 M NaCl, 60 mM Imidazole, pH 7.9).Poly His containing protein could be eluted with Elute buffer (20 mMTris-HCl, 0.5 M NaCl, 1 M Imidazole, pH 7.9) and dialyzes against 1×binding buffer without imidazole.

However, to remove the poly His sequence to yield a native protein, thecolumn was instead washed with 10 times the bed volume with thrombincleavage buffer (20 mM Tris-HCl, pH7.5, 150 mM NaCl, 2.5 mM CaCl₂). Then1 bed volume of biotinylated thrombin solution (1 u/mg of protein,Novagen) was added and the column incubated at room temperatureovernight. Untagged proteins were eluted by thrombin cleavage buffer.The biotinylated thrombins were cleared by steptaviding agarose(supplied in the Novagen Thrombin cleavage kit) using a ratio of 16-μlresin per unit of enzyme. Finally, proteins were dialyzed with 1×binding buffer without imidazole. Uncleaved protein could be recoveredfrom the column as above. The nucleotide and amino acid sequence of theWWP2 recombinant protein are shown in Table VI.

TABLE VI Nucleotide and amino acid sequences for WWP2 recombinantprotein SEQ ID NO: Sequence¹ 23gaattcggcttcgggatccaccATGGATTACAAGGATGACGACGATAAGATGGCATCTGCCAGCTCTAGCCGGGCAGGAGTGGCCCTGCCTTTTGAGAAGTCTCAGCTCACTTTGAAAGTGGTGTCCGCAAAGCCCAAGGTGCATAATCGTCAACCTCGAATTAACTCCTACGTGGAGGTGGCGGTGGATGGACTCCCCAGTGAGACCAAGAAGACTGGGAAGCGCATTGGGAGCTCTGAGCTTCTCTGGAATGAGATCATCATTTTGAATGTCACGGCACAGAGTCATTTAGATTTAAAGGTCTGGAGCTGCCATACCTTGAGAAATGAACTGCTAGGCACCGCATCTGTCAACCTCTCCAACGTCTTGAAGAACAATGGGGGCAAAATGGAGAACATGCAGCTGACCCTGAACCTGCAGACGGAGAACAAAGGCAGCGTTGTCTCAGGCGGAGAGCTGACAATTTTCCTGGACGGGCCAACTGTTGATCTGGGAAATGTGCCTAATGGCAGTGCCCTGACAGATGGATCACAGCTGCCTTCGAGAGACTCCAGTGGAACAGCAGTAGCTCCAGAGAACCGGCACCAGCCCCCCAGCACAAACTGCTTTGGTGGAAGATCCCGGACGCACAGACATTCGGGTGCTTCAGCCAGAACAACCCCAGCAACCGGCGAGCAAAGCCCCGGTGCTCGGAGCCGGCACCGCCAGCCCGTCAAGAACTCAGGCCACAGTGGCTTGGCCAATGGCACAGTGAATGATGAACCCACAACAGCCACTGATCCCGAAGAACCTTCCGTTGTTGGTGTGACGTCCCCACCTGCTGCACCCTTGAGTGTGACCCCGAATCCCAACACGACTTCTCTCCCTGCCCCAGCCACACCGGCTGAAGGAGAGGAACCCAGCACTTCGGGTACACAGCAGCTCCCAGCGGCTGCCCAGGCCCCCGACGCTCTGCCTGCTGGATGGGAACAGCGAGAGCTGCCCAACGGACGTGTCTATTATGTTGACCACAATACCAAGACCACCACCTGGGAGCGGCCCCTTCCTCCAGGCTGGGAAAAACGCACAGATCCCCGAGGCAGGTTTTACTATGTGGATCACAATACTCGGACCACCACCTGGCAGCGTCCGACCGCGGAGTACGTGCGCAACTATGAGCAGTGGCAGTCGCAGCGGAATCAGCTCCAGGGGGCCATGCAGCACTTCAGCCAAAGATTCCTCTACCAGTCTTCGAGTGCTTCGACTGACCATGATCCCCTGGGCCCCCTCCCTCCTGGCTGGGAGAAGAGACAGGACAATGGACGGGTGTATTACGTGAACCATAACACTCGCACGACCCAGTGGGAGGATCCCCGGACCCAGGGGATGATCCAGGAACCAGCTCTGCCCCCAGGATGGGAGATGAAATACACCAGCGAGGGGGTGCGATACTTTGTGGACCACAATACCCGCACCACCACCTTTAAGGATCCTCGCCCGGGGTTTGAGTCGGGGACGAAGCAAGGTTCCCCTGGTGCTTATGACCGCAGTTTTCGGTGGAAGTATCACCAGTTCCGTTTCCTCTGCCATTCAAATGCCCTACCTAGCCACGTGAAGATCAGCGTTTCCAGGCAGACGCTTTTCGAAGATTCCTTCCAACAGATCATGAACATGAAACCCTATGACCTGCGCCGCCGGCTCTACATCATCATGCGTGGCGAGGAGGGCCTGGACTATGGGGGCATCGCCAGAGAGTGGTTTTTCCTCCTGTCTCATGAGGTGCTCAACCCTATGTATTGTTTATTTGAATATGCCGGAAAGAACAATTACTGCCTGCAGATCAACCCCGCCTCCTCCATCAACCCGGACCACCTCACCTACTTTCGCTTTATAGGCAGATTCATCGCCATGGCGCTGTACCATGGAAAGTTCATCGACACGGGCTTCACCCTCCCTTTCTACAAGCGGATGCTCAATAAGAGACCAACCCTGAAAGACCTGGAGTCCATTGACCCTGAGTTCTACAACTCCATTGTCTGGATCAAAGAGAACAACCTGGAAGAATGTGGCCTGGAGCTGTACTTCATCCAGGACATGGAGATACTGGGCAAGGTGACGACCCACGAGCTGAAGGAGGGCGGCGAGAGCATCCGGGTCACAGAGGAGAACAAGGAAGAGTACATCATGCTGCTGACTGACTGGCGTTTCACCCGAGGCGTGGAAGAGCAGACCAAAGCCTTCCTGGATGGCTTCAACGAGGTGGCCCCGCTGGAGTGGCTGCGCTACTTTGACGAGAAAGAGCTGGAGCTGATGCTGTGCGGCATGCAGGAGATAGACATGAGCGACTGGCAGAAGAGCACCATCTACCGGCACTACACCAAGAACAGCAAGCAGATCCAGTGGTTCTGGCAGGTGGTGAAGGAGATGGACAACGAGAAGAGGATCCGGCTGCTGCAGTTTGTCACCGGTACCTGCCGCCTGCCCGTCGGGGGATTTGCCGAACTCATCGGTAGCAACGGACCACAGAAGTTTTGCATTGACAAAGTTGGCAAGGAAACCTGGCTGCCCAGAAGCCACACCTGCTTCAACCGTCTGGATCTTCCACCCTACAAGAGCTACGAACAGCTGAGAGAGAAGCTGCTGTATGCCATTGAGGAGACCGAGGGCTTTGGACAGGAGTAA ctcgag 24MDYKDDDDKMASASSSRAGVALPFEKSQLTLKVVSAKPKVHNRQPRINSYVEVAVDGLPSETKKTGKRIGSSELLWNEIIILNVTAQSHLDLKVWSCHTLRNELLGTASVNLSNVLKNNGGKMENMQLTLNLQTENKGSVVSGGELTIFLDGPTVDLGNVPNGSALTDGSQLPSRDSSGTAVAPENRHQPPSTNCFGGRSRTHRHSGASARTTPATGEQSPGARSRHRQPVKNSGHSGLANGTVNDEPTTATDPEEPSVVGVTSPPAAPLSVTPNPNTTSLPAPATPAEGEEPSTSGTQQLPAAAQAPDALPAGWEQRELPNGRVYYVDHNTKTTTWERPLPPGWEKRTDPRGRFYYVDHNTRTTTWQRPTAEYVRNYEQWQSQRNQLQGAMQHFSQRFLYQSSSASTDHDPLGPLPPGWEKRQDNGRVYYVNHNTRTTQWEDPRTQGMIQEPALPPGWENKYTSEGVRYFVDHNTRTTTFKDPRPGFESGTKQGSPGAYDRSFRWKYHQFRFLCHSNALPSHVKISVSRQTLFEDSFQQIMNMKPYDLRRRLYIIMRGEEGLDYGGIAREWFFLLSHEVLNPMYCLFEYAGKNNYCLQINPASSINPDHLTYFRFIGRFIAMALYHGKFIDTGFTLPFYKRMLNKRPTLKDLESIDPEFYNSIVWIKENNLEECGLELYFIQDMEILGKVTTHELKEGGESIRVTEENKEEYIMLLTDWRFTRGVEEQTKAFLDGFNEVAPLEWLRYFDEKELELMLCGMQEIDMSDWQKSTIYRHYTKNSKQTQWFWQVVKEMDNEKRIRLLQFVTGTCRLPVGGFAELIGSNGPQKFCIDKVGKETWLPRSHTCFNRLDLPPYKSYEQLREKLLYAIEETEGFGQE ¹The upper case nucleotidesequence denotes the recombinant polypeptide coding sequence, whereinthe italicized sequence encodes the FLAG tag epitope. The underlinednucleotide sequences at the 5′- and 3′-termini of the nucleotidesequence correspond to the EcoRI and XhoI restriction site sequences forintroducing the recombinant insert into the pET28b vector.

The complete sequence of the pET28b expression vector that includes theWWP2 recombinant protein is presented below.

SEQ ID NO: 25:TGGCGAATGGGACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGGGGGCTCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCACCTCGACCCCAAAAAACTTGATTAGGGTGATGGTTCACGTAGTGGGCCATCGCCCTGATAGACGGTTTTTCGCCCTTTGACGTTGGAGTCCACGTTCTTTAATAGTGGACTCTTGTTCCAAACTGGAACAACACTCAACCCTATCTCGGTCTATTCTTTTGATTTATAAGGGATTTTGCCGATTTCGGCCTATTGGTTAAAAAATGAGCTGATTTAACAAAAATTTAACGCGAATTTTAACAAAATATTAACGTTTACAATTTCAGGTGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAATTAATTCTTAGAAAAACTCATCGAGCATCAAATGAAACTGCAATTTATTCATATCAGGATTATCAATACCATATTTTTGAAAAAGCCGTTTCTGTAATGAAGGAGAAAACTCACCGAGGCAGTTCCATAGGATGGCAAGATCCTGGTATCGGTCTGCGATTCCGACTCGTCCAACATCAATACAACCTATTAATTTCCCCTCGTCAAAAATAAGGTTATCAAGTGAGAAATCACCATGAGTGACGACTGAATCCGGTGAGAATGGCAAAAGTTTATGCATTTCTTTCCAGACTTGTTCAACAGGCCAGCCATTACGCTCGTCATCAAAATCACTCGCATCAACCAAACCGTTATTCATTCGTGATTGCGCCTGAGCGAGACGAAATACGCGATCGCTGTTAAAAGGACAATTACAAACAGGAATCGAATGCAACCGGCGCAGGAACACTGCCAGCGCATCAACAATATTTTCACCTGAATCAGGATATTCTTCTAATACCTGGAATGCTGTTTTCCCGGGGATCGCAGTGGTGAGTAACCATGCATCATCAGGAGTACGGATAAAATGCTTGATGGTCGGAAGAGGCATAAATTCCGTCAGCCAGTTTAGTCTGACCATCTCATCTGTAACATCATTGGCAACGCTACCTTTGCCATGTTTCAGAAACAACTCTGGCGCATCGGGCTTCCCATACAATCGATAGATTGTCGCACCTGATTGCCCGACATTATCGCGAGCCCATTTATACCCATATAAATCAGCATCCATGTTGGAATTTAATCGCGGCCTAGAGCAAGACGTTTCCCGTTGAATATGGCTCATAACACCCCTTGTATTACTGTTTATGTAAGCAGACAGTTTTATTGTTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTCCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGAGGAAGCGGAAGAGCGCCTGATGCGGTATTTTCTCCTTACGCATCTGTGCGGTATTTCACACCGCATATATGGTGCACTCTCAGTACAATCTGCTCTGATGCCGCATAGTTAAGCCAGTATACACTCCGCTATCGCTACGTGACTGGGTCATGGCTGCGCCCCGACACCCGCCAACACCCGCTGACGCGCCCTGACGGGCTTGTCTGCTCCCGGCATCCGCTTACAGACAAGCTGTGACCGTCTCCGGGAGCTGCATGTGTCAGAGGTTTTCACCGTCATCACCGAAACGCGCGAGGCAGCTGCGGTAAAGCTCATCAGCGTGGTCGTGAAGCGATTCACAGATGTCTGCCTGTTCATCCGCGTCCAGCTCGTTGAGTTTCTCCAGAAGCGTTAATGTCTGGCTTCTGATAAAGCGGGCCATGTTAAGGGCGGTTTTTTCCTGTTTGGTCACTGATGCCTCCGTGTAAGGGGGATTTCTGTTCATGGGGGTAATGATACCGATGAAACGAGAGAGGATGCTCACGATACGGGTTACTGATGATGAACATGCCCGGTTACTGGAACGTTGTGAGGGTAAACAACTGGCGGTATGGATGCGGCGGGACCAGAGAAAAATCACTCAGGGTCAATGCCAGCGCTTCGTTAATACAGATGTAGGTGTTCCACAGGGTAGCCAGCAGCATCCTGCGATGCAGATCCGGAACATAATGGTGCAGGGCGCTGACTTCCGCGTTTCCAGACTTTACGAAACACGGAAACCGAAGACCATTCATGTTGTTGCTCAGGTCGCAGACGTTTTGCAGCAGCAGTCGCTTCACGTTCGCTCGCGTATCGGTGATTCATTCTGCTAACCAGTAAGGCAACCCCGCCAGCCTAGCCGGGTCCTCAACGACAGGAGCACGATCATGCGCACCCGTGGGGCCGCCATGCCGGCGATAATGGCCTGCTTCTCGCCGAAACGTTTGGTGGCGGGACCAGTGACGAAGGCTTGAGCGAGGGCGTGCAAGATTCCGAATACCGCAAGCGACAGGCCGATCATCGTCGCGCTCCAGCGAAAGCGGTCCTCGCCGAAAATGACCCAGAGCGCTGCCGGCACCTGTCCTACGAGTTGCATGATAAAGAAGACAGTCATAAGTGCGGCGACGATAGTCATGCCCCGCGCCCACCGGAAGGAGCTGACTGGGTTGAAGGCTCTCAAGGGCATCGGTCGAGATCCCGGTGCCTAATGAGTGAGCTAACTTACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCCAGGGTGGTTTTTCTTTTCACCAGTGAGACGGGCAACAGCTGATTGCCCTTCACCGCCTGGCCCTGAGAGAGTTGCAGCAAGCGGTCCACGCTGGTTTGCCCCAGCAGGCGAAAATCCTGTTTGATGGTGGTTAACGGCGGGATATAACATGAGCTGTCTTCGGTATCGTCGTATCCCACTACCGAGATATCCGCACCAACGCGCAGCCCGGACTCGGTAATGGCGCGCATTGCGCCCAGCGCCATCTGATCGTTGGCAACCAGCATCGCAGTGGGAACGATGCCCTCATTCAGCATTTGCATGGTTTGTTGAAAACCGGACATGGCACTCCAGTCGCCTTCCCGTTCCGCTATCGGCTGAATTTGATTGCGAGTGAGATATTTATGCCAGCCAGCCAGACGCAGACGCGCCGAGACAGAACTTAATGGGCCCGCTAACAGCGCGATTTGCTGGTGACCCAATGCGACCAGATGCTCCACGCCCAGTCGCGTACCGTCTTCATGGGAGAAAATAATACTGTTGATGGGTGTCTGGTCAGAGACATCAAGAAATAACGCCGGAACATTAGTGCAGGCAGCTTCCACAGCAATGGCATCCTGGTCATCCAGCGGATAGTTAATGATCAGCCCACTGACGCGTTGCGCGAGAAGATTGTGCACCGCCGCTTTACAGGCTTCGACGCCGCTTCGTTCTACCATCGACACCACCACGCTGGCACCCAGTTGATCGGCGCGAGATTTAATCGCCGCGACAATTTGCGACGGCGCGTGCAGGGCCAGACTGGAGGTGGCAACGCCAATCAGCAACGACTGTTTGCCCGCCAGTTGTTGTGCCACGCGGTTGGGAATGTAATTCAGCTCCGCCATCGCCGCTTCCACTTTTTCCCGCGTTTTCGCAGAAACGTGGCTGGCCTGGTTCACCACGCGGGAAACGGTCTGATAAGAGACACCGGCATACTCTGCGACATCGTATAACGTTACTGGTTTCACATTCACCACCCTGAATTGACTCTCTTCCGGGCGCTATCATGCCATACCGCGAAAGGTTTTGCGCCATTCGATGGTGTCCGGGATCTCGACGCTCTCCCTTATGCGACTCCTGCATTAGGAAGCAGCCCAGTAGTAGGTTGAGGCCGTTGAGCACCGCCGCCGCAAGGAATGGTGCATGCAAGGAGATGGCGCCCAACAGTCCCCCGGCCACGGGGCCTGCCACCATACCCACGCCGAAACAAGCGCTCATGAGCCCGAAGTGGCGAGCCCGATCTTCCCCATCGGTGATGTCGGCGATATAGGCGCCAGCAACCGCACCTGTGGCGCCGGTGATGCCGGCCACGATGCGTCCGGCGTAGAGGATCGAGATCTCGATCCCGCGAAATTAATACGACTCACTATAGGGGAATTGTGAGCGGATAACAATTCCCCTCTAGAAATAATTTTGTTTAACTTTAAGAAGGAGATATACCATGGGCAGCAGCCATCATCATCATCATCACAGCAGCGGCCTGGTGCCGCGCGGCAGCCATATGGCTAGCATGACTGGTGGACAGCAAATGGGTCGCGGATCCGAATTCggcttcgggatccaccATGGATTACAAGGATGACGACGATAAGatggcatctgccagctctagccgggcaggagtggccctgccttttgagaagtctcagctcactttgaaagtggtgtccgcaaagcccaaggtgcataatcgtcaacctcgaattaactcctacgtggaggtggcggtggatggactccccagtgagaccaagaagactgggaagcgcattgggagctctgagcttctctggaatgagatcatcattttgaatgtcacggcacagagtcatttagatttaaaggtctggagctgccataccttgagaaatgaactgctaggcaccgcatctgtcaacctctccaacgtcttgaagaacaatgggggcaaaatggagaacatgcagctgaccctgaacctgcagacggagaacaaaggcagcgttgtctcaggcggagagctgacaattttcctggacgggccaactgttgatctgggaaatgtgcctaatggcagtgccctgacagatggatcacagctgccttcgagagactccagtggaacagcagtagctccagagaaccggcaccagccccccagcacaaactgctttggtggaagatcccggacgcacagacattcgggtgcttcagccagaacaaccccagcaaccggcgagcaaagccccggtgctcggagccggcaccgccagcccgtcaagaactcaggccacagtggcttggccaatggcacagtgaatgatgaacccacaacagccactgatcccgaagaaccttccgttgttggtgtgacgtccccacctgctgcacccttgagtgtgaccccgaatcccaacacgacttctctccctgccccagccacaccggctgaaggagaggaacccagcacttcgggtacacagcagctcccagcggctgcccaggcccccgacgctctgcctgctggatgggaacagcgagagctgcccaacggacgtgtctattatgttgaccacaataccaagaccaccacctgggagcggccccttcctccaggctgggaaaaacgcacagatccccgaggcaggttttactatgtggatcacaatactcggaccaccacctggcagcgtccgaccgcggagtacgtgcgcaactatgagcagtggcagtcgcagcggaatcagctccagggggccatgcagcacttcagccaaagattcctctaccagtcttcgagtgcttcgactgaccatgatcccctgggccccctccctcctggctgggagaagagacaggacaatggacgggtgtattacgtgaaccataacactcgcacgacccagtgggaggatccccggacccaggggatgatccaggaaccagctctgcccccaggatgggagatgaaatacaccagcgagggggtgcgatactttgtggaccacaatacccgcaccaccacctttaaggatcctcgcccggggtttgagtcggggacgaagcaaggttcccctggtgcttatgaccgcagttttcggtggaagtatcaccagttccgtttcctctgccattcaaatgccctacctagccacgtgaagatcagcgtttccaggcagacgcttttcgaagattccttccaacagatcatgaacatgaaaccctatgacctgcgccgccggctctacatcatcatgcgtggcgaggagggcctggactatgggggcatcgccagagagtggtttttcctcctgtctcatgaggtgctcaaccctatgtattgtttatttgaatatgccggaaagaacaattactgcctgcagatcaaccccgcctcctccatcaacccggaccacctcacctactttcgctttataggcagattcatcgccatggcgctgtaccatggaaagttcatcgacacgggcttcaccctccctttctacaagcggatgctcaataagagaccaaccctgaaagacctggagtccattgaccctgagttctacaactccattgtctggatcaaagagaacaacctggaagaatgtggcctggagctgtacttcatccaggacatggagatactgggcaaggtgacgacccacgagctgaaggagggcggcgagagcatccgggtcacagaggagaacaaggaagagtacatcatgctgctgactgactggcgtttcacccgaggcgtggaagagcagaccaaagccttcctggatggcttcaacgaggtggccccgctggagtggctgcgctactttgacgagaaagagctggagctgatgctgtgcggcatgcaggagatagacatgagcgactggcagaagagcaccatctaccggcactacaccaagaacagcaagcagatccagtggttctggcaggtggtgaaggagatggacaacgagaagaggatccggctgctgcagtttgtcaccggtacctgccgcctgcccgtcgggggatttgccgaactcatcggtagcaacggaccacagaagttttgcattgacaaagttggcaaggaaacctggctgcccagaagccacacctgcttcaaccgtctggatcttccaccctacaagagctacgaacagctgagagagaagctgctgtatgccattgaggagaccgagggctttggacaggagtaaCTCGAGCACCACCACCACCACCACTGAGATCCGGCTGCTAACAAAGCCCGAAAGGAAGCTGAGTTGGCTGCTGCCACCGCTGAGCAATAACTAGCATAACCCCTTGGGGCCTCTAAACGGGTCTTGAGGGGTTTTTTGCTGAAAGGAGGAACTATATCCGGATwherein the bold font denote the locations of the initiator andterminator codons for the recombinant peptide pro-form; the upper caseletters denote pET28b vector sequences; the lower case letter denote theWWP2 coding sequences, the single-underlined sequences are the poly-Hiscoding sequences, the double-underlined sequences encode the Thrombincleavage site, the italicized font includes the FLAG-tag codingsequences in frame with the WWP2 coding sequences as configured in theexpression vector, pET28b (Novagen).

Construction and Preparation of WWP1 Protein.

The WWP1 encoding gene was amplified by PCR reaction fromFLAG-WWP1_pCNA3.1 construct and was ligated to pET28b(+) His-tag plasmid(Novagen). His-tagged WWP1 plasmids were transformed into BL21 DE3cells. Protein expression and purification were followed the procedureof WWP2 protein preparation. The WWP1 encoding gene was amplified by PCRreaction using Deep Vent polymerase (NEB) from the FLAG-WWP1_pCNA3.1construct using the oligodeoxynucleotides5′-ATAGGATTCATGGCCACTGCTTCACCAAGGTCT-3′ forward primer (SEQ ID NO:26)and 5′-ATAGCGGCCGCTCATTCTTGTCCAAATCCCTCTGT-3′ reverse primer (SEQ IDNO:27) and was ligated to pET28b(+) His-tag plasmid between the EcoR1and Not cloning sites. The nucleotide and amino acid sequences for WWP1recombinant protein are illustrated in Table VII.

TABLE VII Nucleotide and amino acid sequences for WWP1recombinant protein¹ SEQ ID NO: Sequence² 28gaattcATGGCCACTGCTTCACCAAGGTCTGATACTAGTAATAACCACAGTGGAAGGTTGCAGTTACAGGTAACTGTTTCTAGTGCCAAACTTAAAAGAAAAAAGAACTGGTTCGGAACAGCAATATATACAGAAGTAGTTGTAGATGGAGAAATTACGAAAACAGCAAAATCCAGTAGTTCTTCTAATCCAAAATGGGATGAACAGCTAACTGTAAATGTTACGCCACAGACTACATTGGAATTTCAAGTTTGGAGCCATCGCACTTTAAAAGCAGATGCTTTATTAGGAAAAGCAACGATAGATTTGAAACAAGCTCTGTTGATACACAATAGAAAATTGGAAAGAGTGAAAGAACAATTAAAACTTTCCTTGGAAAACAAGAATGGCATAGCACAAACTGGTGAATTGACAGTTGTGCTTGATGGATTGGTGATTGAGCAAGAAAATATAACAAACTGCAGCTCATCTCCAACCATAGAAATACAGGAAAATGGTGATGCCTTACATGAAAATGGAGAGCCTTCAGCAAGGACAACTGCCAGGTTGGCTGTTGAAGGCACGAATGGAATAGATAATCATGTACCTACAAGCACTCTAGTCCAAAACTCATGCTGCTCGTATGTAGTTAATGGAGACAACACACCTTCATCTCCGTCTCAGGTTGCTGCCAGACCCAAAAATACACCAGCTCCAAAACCACTCGCATCTGAGCCTGCCGATGACACTGTTAATGGAGAATCATCCTCATTTGCACCAACTGATAATGCGTCTGTCACGGGTACTCCAGTAGTGTCTGAAGAAAATGCCTTGTCTCCAAATTGCACTAGTACTACTGTTGAAGATCCTCCAGTTCAAGAAATACTGACTTCCTCAGAAAACAATGAATGTATTCCTTCTACCAGTGCAGAATTGGAATCTGAAGCTAGAAGTATATTAGAGCCTGACACCTCTAATTCTAGAAGTAGTTCTGCTTTTGAAGCAGCCAAATCAAGACAGCCAGATGGGTGTATGGATCCTGTACGGCAGCAGTCTGGGAATGCCAACACAGAAACCTTGCCATCAGGGTGGGAACAAAGAAAAGATCCTCATGGTAGAACCTATTATGTGGATCATAATACTCGAACTACCACATGGGAGAGACCACAACCTTTACCTCCAGGTTGGGAAAGAAGAGTTGATGATCGTAGAAGAGTTTATTATGTGGATCATAACACCAGAACAACAACGTGGCAGCGGCCTACCATGGAATCTGTCCGAAATTTTGAACAGTGGCAATCTCAGCGGAACCAATTGCAGGGAGCTATGCAACAGTTTAACCAACGATACCTCTATTCGGCTTCAATGTTAGCTGCAGAAAATGACCCTTATGGACCTTTGCCACCAGGCTGGGAAAAAAGAGTGGATTCAACAGACAGGGTTTACTTTGTGAATCATAACACAAAAACAACCCAGTGGGAAGATCCAAGAACTCAAGGCTTACAGAATGAAGAACCCCTGCCAGAAGGCTGGGAAATTAGATATACTCGTGAAGGTGTAAGGTACTTTGTTGATCATAACACAAGAACAACAACATTCAAAGATCCTCGCAATGGGAAGTCATCTGTAACTAAAGGTGGTCCACAAATTGCTTATGAACGCGGCTTTAGGTGGAAGCTTGCTCACTTCCGTTATTTGTGCCAGTCTAATGCACTACCTAGTCATGTAAAGATCAATGTGTCCCGGCAGACATTGTTTGAAGATTCCTTCCAACAGATTATGGCATTAAAACCCTATGACTTGAGGAGGCGCTTATATGTAATATTTAGAGGAGAAGAAGGACTTGATTATGGTGGCCTAGCGAGAGAATGGTTTTTCTTGCTTTCACATGAAGTTTTGAACCCAATGTATTGCTTATTTGAGTATGCGGGCAAGAACAACTATTGTCTGCAGATAAATCCAGCATCAACCATTAATCCAGACCATCTTTCATACTTCTGTTTCATTGGTCGTTTTATTGCCATGGCACTATTTCATGGAAAGTTTATCGATACTGGTTTCTCTTTACCATTCTACAAGCGTATGTTAAGTAAAAAACTTACTATTAAGGATTTGGAATCTATTGATACTGAATTTTATAACTCCCTTATCTGGATAAGAGATAACAACATTGAAGAATGTGGCTTAGAAATGTACTTTTCTGTTGACATGGAGATTTTGGGAAAAGTTACTTCACATGACCTGAAGTTGGGAGGTTCCAATATTCTGGTGACTGAGGAGAACAAAGATGAATATATTGGTTTAATGACAGAATGGCGTTTTTCTCGAGGAGTACAAGAACAGACCAAAGCTTTCCTTGATGGTTTTAATGAAGTTGTTCCTCTTCAGTGGCTACAGTACTTCGATGAAAAAGAATTAGAGGTTATGTTGTGTGGCATGCAGGAGGTTGACTTGGCAGATTGGCAGAGAAATACTGTTTATCGACATTATACAAGAAACAGCAAGCAAATCATTTGGTTTTGGCAGTTTGTGAAAGAGACAGACAATGAAGTAAGAATGCGACTATTGCAGTTCGTCACTGGAACCTGCCGTTTACCTCTAGGAGGATTTGCTGAGCTCATGGGAAGTAATGGGCCTCAAAAGTTTTGCATTGAAAAAGTTGGCAAAGACACTTGGTTACCAAGAAGCCATACATGTTTTAATCGCTTGGATCTACCACCATATAAGAGTTATGAACAACTAAAGGAAAAACTTCTTTTTGCAATAGAAGAGACAGAGGGATTTG GACAAGAATGAgcggccgc 29MATASPRSDTSNNHSGRLQLQVTVSSAKLKRKKNWFGTAIYTEVVVDGEITKTAKSSSSSNPKWDEQLTVNVTPQTTLEFQVWSHRTLKADALLGKATIDLKQALLIHNRKLERVKEQLKLSLENKNGIAQTGELTVVLDGLVIEQENITNCSSSPTIEIQENGDALHENGEPSARTTARLAVEGTNGIDNHVPTSTLVQNSCCSYVVNGDNTPSSPSQVAARPKNTPAPKPLASEPADDTVNGESSSFAPTDNASVTGTPVVSEENALSPNCTSTTVEDPPVQEILTSSENNECIPSTSAELESEARSILEPDTSNSRSSSAFEAAKSRQPDGCMDPVRQQSGNANTETLPSGWEQRKDPHGRTYYVDHNTRTTTWERPQPLPPGWERRVDDRRRVYYVDHNTRTTTWQRPTMESVRNFEQWQSQRNQLQGAMQQFNQRYLYSASMLAAENDPYGPLPPGWEKRVDSTDRVYFVNHNTKTTQWEDPRTQGLQNEEPLPEGWEIRYTREGVRYFVDHNTRTTTFKDPRNGKSSVTKGGPQIAYERGFRWKLAHFRYLCQSNALPSHVKINVSRQTLFEDSFQQIMALKPYDLRRRLYVIFRGEEGLDYGGLAREWFFLLSHEVLNPMYCLFEYAGKNNYCLQINPASTINPDHLSYFCFIGRFIAMALFHGKFIDTGFSLPFYKRMLSKKLTIKDLESIDTEFYNSLIWIRDNNIEECGLEMYFSVDMEILGKVTSHDLKLGGSNILVTEENKDEYIGLMTEWRFSRGVQEQTKAFLDGFNEVVPLQWLQYFDEKELEVMLCGMQEVDLADWQRNTVYRHYTRNSKQIIWFWQFVKETDNEVRMRLLQFVTGTCRLPLGGFAELMGSNGPQKFCIEKVGKDTWLPRSHTCFNRLDLPPYKSYEQLKEKLLFAIEETEGFGQE ¹Recombinant protein illustrated without theHis-tag sequence present (cleaved off). ²The upper case nucleotidesequence denotes the recombinant polypeptide coding sequence. Theunderlined nucleotide sequences at the 5′- and 3′-termini of thenucleotide sequence correspond to the EcoRI and NotI restriction sitesequences for introducing the recombinant insert into the pET28b vector.

The complete sequence of the pET28b expression vector that includes theWWP1 recombinant protein is presented below.

SEQ ID NO: 30:TGGCGAATGGGACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGGGGGCTCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCACCTCGACCCCAAAAAACTTGATTAGGGTGATGGTTCACGTAGTGGGCCATCGCCCTGATAGACGGTTTTTCGCCCTTTGACGTTGGAGTCCACGTTCTTTAATAGTGGACTCTTGTTCCAAACTGGAACAACACTCAACCCTATCTCGGTCTATTCTTTTGATTTATAAGGGATTTTGCCGATTTCGGCCTATTGGTTAAAAAATGAGCTGATTTAACAAAAATTTAACGCGAATTTTAACAAAATATTAACGTTTACAATTTCAGGTGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAATTAATTCTTAGAAAAACTCATCGAGCATCAAATGAAACTGCAATTTATTCATATCAGGATTATCAATACCATATTTTTGAAAAAGCCGTTTCTGTAATGAAGGAGAAAACTCACCGAGGCAGTTCCATAGGATGGCAAGATCCTGGTATCGGTCTGCGATTCCGACTCGTCCAACATCAATACAACCTATTAATTTCCCCTCGTCAAAAATAAGGTTATCAAGTGAGAAATCACCATGAGTGACGACTGAATCCGGTGAGAATGGCAAAAGTTTATGCATTTCTTTCCAGACTTGTTCAACAGGCCAGCCATTACGCTCGTCATCAAAATCACTCGCATCAACCAAACCGTTATTCATTCGTGATTGCGCCTGAGCGAGACGAAATACGCGATCGCTGTTAAAAGGACAATTACAAACAGGAATCGAATGCAACCGGCGCAGGAACACTGCCAGCGCATCAACAATATTTTCACCTGAATCAGGATATTCTTCTAATACCTGGAATGCTGTTTTCCCGGGGATCGCAGTGGTGAGTAACCATGCATCATCAGGAGTACGGATAAAATGCTTGATGGTCGGAAGAGGCATAAATTCCGTCAGCCAGTTTAGTCTGACCATCTCATCTGTAACATCATTGGCAACGCTACCTTTGCCATGTTTCAGAAACAACTCTGGCGCATCGGGCTTCCCATACAATCGATAGATTGTCGCACCTGATTGCCCGACATTATCGCGAGCCCATTTATACCCATATAAATCAGCATCCATGTTGGAATTTAATCGCGGCCTAGAGCAAGACGTTTCCCGTTGAATATGGCTCATAACACCCCTTGTATTACTGTTTATGTAAGCAGACAGTTTTATTGTTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTCCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGAGGAAGCGGAAGAGCGCCTGATGCGGTATTTTCTCCTTACGCATCTGTGCGGTATTTCACACCGCATATATGGTGCACTCTCAGTACAATCTGCTCTGATGCCGCATAGTTAAGCCAGTATACACTCCGCTATCGCTACGTGACTGGGTCATGGCTGCGCCCCGACACCCGCCAACACCCGCTGACGCGCCCTGACGGGCTTGTCTGCTCCCGGCATCCGCTTACAGACAAGCTGTGACCGTCTCCGGGAGCTGCATGTGTCAGAGGTTTTCACCGTCATCACCGAAACGCGCGAGGCAGCTGCGGTAAAGCTCATCAGCGTGGTCGTGAAGCGATTCACAGATGTCTGCCTGTTCATCCGCGTCCAGCTCGTTGAGTTTCTCCAGAAGCGTTAATGTCTGGCTTCTGATAAAGCGGGCCATGTTAAGGGCGGTTTTTTCCTGTTTGGTCACTGATGCCTCCGTGTAAGGGGGATTTCTGTTCATGGGGGTAATGATACCGATGAAACGAGAGAGGATGCTCACGATACGGGTTACTGATGATGAACATGCCCGGTTACTGGAACGTTGTGAGGGTAAACAACTGGCGGTATGGATGCGGCGGGACCAGAGAAAAATCACTCAGGGTCAATGCCAGCGCTTCGTTAATACAGATGTAGGTGTTCCACAGGGTAGCCAGCAGCATCCTGCGATGCAGATCCGGAACATAATGGTGCAGGGCGCTGACTTCCGCGTTTCCAGACTTTACGAAACACGGAAACCGAAGACCATTCATGTTGTTGCTCAGGTCGCAGACGTTTTGCAGCAGCAGTCGCTTCACGTTCGCTCGCGTATCGGTGATTCATTCTGCTAACCAGTAAGGCAACCCCGCCAGCCTAGCCGGGTCCTCAACGACAGGAGCACGATCATGCGCACCCGTGGGGCCGCCATGCCGGCGATAATGGCCTGCTTCTCGCCGAAACGTTTGGTGGCGGGACCAGTGACGAAGGCTTGAGCGAGGGCGTGCAAGATTCCGAATACCGCAAGCGACAGGCCGATCATCGTCGCGCTCCAGCGAAAGCGGTCCTCGCCGAAAATGACCCAGAGCGCTGCCGGCACCTGTCCTACGAGTTGCATGATAAAGAAGACAGTCATAAGTGCGGCGACGATAGTCATGCCCCGCGCCCACCGGAAGGAGCTGACTGGGTTGAAGGCTCTCAAGGGCATCGGTCGAGATCCCGGTGCCTAATGAGTGAGCTAACTTACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCCAGGGTGGTTTTTCTTTTCACCAGTGAGACGGGCAACAGCTGATTGCCCTTCACCGCCTGGCCCTGAGAGAGTTGCAGCAAGCGGTCCACGCTGGTTTGCCCCAGCAGGCGAAAATCCTGTTTGATGGTGGTTAACGGCGGGATATAACATGAGCTGTCTTCGGTATCGTCGTATCCCACTACCGAGATATCCGCACCAACGCGCAGCCCGGACTCGGTAATGGCGCGCATTGCGCCCAGCGCCATCTGATCGTTGGCAACCAGCATCGCAGTGGGAACGATGCCCTCATTCAGCATTTGCATGGTTTGTTGAAAACCGGACATGGCACTCCAGTCGCCTTCCCGTTCCGCTATCGGCTGAATTTGATTGCGAGTGAGATATTTATGCCAGCCAGCCAGACGCAGACGCGCCGAGACAGAACTTAATGGGCCCGCTAACAGCGCGATTTGCTGGTGACCCAATGCGACCAGATGCTCCACGCCCAGTCGCGTACCGTCTTCATGGGAGAAAATAATACTGTTGATGGGTGTCTGGTCAGAGACATCAAGAAATAACGCCGGAACATTAGTGCAGGCAGCTTCCACAGCAATGGCATCCTGGTCATCCAGCGGATAGTTAATGATCAGCCCACTGACGCGTTGCGCGAGAAGATTGTGCACCGCCGCTTTACAGGCTTCGACGCCGCTTCGTTCTACCATCGACACCACCACGCTGGCACCCAGTTGATCGGCGCGAGATTTAATCGCCGCGACAATTTGCGACGGCGCGTGCAGGGCCAGACTGGAGGTGGCAACGCCAATCAGCAACGACTGTTTGCCCGCCAGTTGTTGTGCCACGCGGTTGGGAATGTAATTCAGCTCCGCCATCGCCGCTTCCACTTTTTCCCGCGTTTTCGCAGAAACGTGGCTGGCCTGGTTCACCACGCGGGAAACGGTCTGATAAGAGACACCGGCATACTCTGCGACATCGTATAACGTTACTGGTTTCACATTCACCACCCTGAATTGACTCTCTTCCGGGCGCTATCATGCCATACCGCGAAAGGTTTTGCGCCATTCGATGGTGTCCGGGATCTCGACGCTCTCCCTTATGCGACTCCTGCATTAGGAAGCAGCCCAGTAGTAGGTTGAGGCCGTTGAGCACCGCCGCCGCAAGGAATGGTGCATGCAAGGAGATGGCGCCCAACAGTCCCCCGGCCACGGGGCCTGCCACCATACCCACGCCGAAACAAGCGCTCATGAGCCCGAAGTGGCGAGCCCGATCTTCCCCATCGGTGATGTCGGCGATATAGGCGCCAGCAACCGCACCTGTGGCGCCGGTGATGCCGGCCACGATGCGTCCGGCGTAGAGGATCGAGATCTCGATCCCGCGAAATTAATACGACTCACTATAGGGGAATTGTGAGCGGATAACAATTCCCCTCTAGAAATAATTTTGTTTAACTTTAAGAAGGAGATATACCATGGGCAGCAGCCATCATCATCATCATCACAGCAGCGGCCTGGTGCCGCGCGGCAGCCATATGGCTAGCATGACTGGTGGACAGCAAATGGGTCGCGGATCCGAATTCatggccactgcttcaccaaggtctgatactagtaataaccacagtggaaggttgcagttacaggtaactgtttctagtgccaaacttaaaagaaaaaagaactggttcggaacagcaatatatacagaagtagttgtagatggagaaattacgaaaacagcaaaatccagtagttcttctaatccaaaatgggatgaacagctaactgtaaatgttacgccacagactacattggaatttcaagtttggagccatcgcactttaaaagcagatgctttattaggaaaagcaacgatagatttgaaacaagctctgttgatacacaatagaaaattggaaagagtgaaagaacaattaaaactttccttggaaaacaagaatggcatagcacaaactggtgaattgacagttgtgcttgatggattggtgattgagcaagaaaatataacaaactgcagctcatctccaaccatagaaatacaggaaaatggtgatgccttacatgaaaatggagagccttcagcaaggacaactgccaggttggctgttgaaggcacgaatggaatagataatcatgtacctacaagcactctagtccaaaactcatgctgctcgtatgtagttaatggagacaacacaccttcatctccgtctcaggttgctgccagacccaaaaatacaccagctccaaaaccactcgcatctgagcctgccgatgacactgttaatggagaatcatcctcatttgcaccaactgataatgcgtctgtcacgggtactccagtagtgtctgaagaaaatgccttgtctccaaattgcactagtactactgttgaagatcctccagttcaagaaatactgacttcctcagaaaacaatgaatgtattccttctaccagtgcagaattggaatctgaagctagaagtatattagagcctgacacctctaattctagaagtagttctgcttttgaagcagccaaatcaagacagccagatgggtgtatggatcctgtacggcagcagtctgggaatgccaacacagaaaccttgccatcagggtgggaacaaagaaaagatcctcatggtagaacctattatgtggatcataatactcgaactaccacatgggagagaccacaacctttacctccaggttgggaaagaagagttgatgatcgtagaagagtttattatgtggatcataacaccagaacaacaacgtggcagcggcctaccatggaatctgtccgaaattttgaacagtggcaatctcagcggaaccaattgcagggagctatgcaacagtttaaccaacgatacctctattcggcttcaatgttagctgcagaaaatgacccttatggacctttgccaccaggctgggaaaaaagagtggattcaacagacagggtttactttgtgaatcataacacaaaaacaacccagtgggaagatccaagaactcaaggcttacagaatgaagaacccctgccagaaggctgggaaattagatatactcgtgaaggtgtaaggtactttgttgatcataacacaagaacaacaacattcaaagatcctcgcaatgggaagtcatctgtaactaaaggtggtccacaaattgcttatgaacgcggctttaggtggaagcttgctcacttccgttatttgtgccagtctaatgcactacctagtcatgtaaagatcaatgtgtcccggcagacattgtttgaagattccttccaacagattatggcattaaaaccctatgacttgaggaggcgcttatatgtaatatttagaggagaagaaggacttgattatggtggcctagcgagagaatggtttttcttgctttcacatgaagttttgaacccaatgtattgcttatttgagtatgcgggcaagaacaactattgtctgcagataaatccagcatcaaccattaatccagaccatctttcatacttctgtttcattggtcgttttattgccatggcactatttcatggaaagtttatcgatactggtttctctttaccattctacaagcgtatgttaagtaaaaaacttactattaaggatttggaatctattgatactgaattttataactcccttatctggataagagataacaacattgaagaatgtggcttagaaatgtacttttctgttgacatggagattttgggaaaagttacttcacatgacctgaagttgggaggttccaatattctggtgactgaggagaacaaagatgaatatattggtttaatgacagaatggcgtttttctcgaggagtacaagaacagaccaaagctttccttgatggttttaatgaagttgttcctcttcagtggctacagtacttcgatgaaaaagaattagaggttatgttgtgtggcatgcaggaggttgacttggcagattggcagagaaatactgtttatcgacattatacaagaaacagcaagcaaatcatttggttttggcagtttgtgaaagagacagacaatgaagtaagaatgcgactattgcagttcgtcactggaacctgccgtttacctctaggaggatttgctgagctcatgggaagtaatgggcctcaaaagttttgcattgaaaaagttggcaaagacacttggttaccaagaagccatacatgttttaatcgcttggatctaccaccatataagagttatgaacaactaaaggaaaaacttctttttgcaatagaagagacagagggatttggacaagaatgaGCGGCCGCACTCGAGCACCACCACCACCACCACTGAGATCCGGCTGCTAACAAAGCCCGAAAGGAAGCTGAGTTGGCTGCTGCCACCGCTGAGCAATAACTAGCATAACCCCTTGGGGCCTCTAAACGGGTCTTGAGGGGTTTTTTGCTGAAAGGAGGAACTATATCCGGATwherein the bold font denote the locations of the initiator andterminator codons for the recombinant peptide pro-form; the upper caseletters denote pET28b vector sequences; the lower case letter denote theWWP1 coding sequences, the italicized font includes the leader peptidecoding sequence that includes a polyhistidine motif and thrombincleavage site derived from the expression vector, pET28b (Novagen).

Example 2. Screening for Small Molecule Compounds that Disrupt theBinding of Nedd4 Proteins to the PY Motif

Fluorescent Polarization Assay (FPA) to Detect Binding of the Nedd4Proteins WWP1/2 to Peptides Containing the PY L-Domain Motif Sequences:

The FPA uses a fluorescein labeled peptide (FTIC-peptide) probe withthree tandem PY motif binding units(FITC-ATASAPPPYVGSGGGATASAPPPYVGSGGGATASAPPPYVGSGGGRRR-OH, Biosynthesis,TX (SEQ ID NO: 18); (SEQ ID NO: 17 corresponds to SEQ ID NO: 18 withoutthe N-terminal FITC moiety) derived from the p2 region of aviansarcoma/leucosis virus (ASLV) gag gene. With the tandem probe, a Kd of0.2 μM was obtained from WWP2 protein dose-response study with highestprotein concentration at 19.6 μM and FITC-peptide probe concentration of5 nM. In contrast, Kd value for the probe with a single binding unitis >15 mM. Screens for compounds that disrupt the binding of theFTIC-peptide and WWP2 (SEQ ID NO:24) were carried out in 384-well plateformat with 25 ml assay volume at ˜0.5 mM WWP2. Non-binding solid blackplates (Corning Costar 3575) were used. The assay buffer contained 20 mMTris and 150 mM NaCl at pH 7.9. FP was measured on either a BiotekSynergy4 or an Analyst GT plate reader. Data analysis was performedusing the in-house software excelHTS. The typical Z value of a screenplate was greater than 0.7. Each assay plate included 16 negativecontrol wells containing protein-probe mix but no compounds and 16 wellscontaining only probe. The difference in FP values of these wells wasused as 100% readout signal to calculate the percentage of inhibition ofa compound well.

The FTIC-labeled probe was premixed at a concentration of 20 nM withWWP1/2 protein at 0.5 mM. Then 25 ml of the protein-probe mix was addedto assay plates by using a ViaFill dispenser. A Labcyte Echo550 acoustictransfer robot was used to transfer compounds (30 to 100 nl, equal to 10to 50 mM) to the assay plates. The plates were shaken to ensure properassay mix. Then FP was measured on an Analyst GT plate reader equippedwith a plate stacker so that data of multiple plates were recorded in asingle file to facilitate data reduction and analysis. The first readwas performed one hour after compound addition then the plates weresealed and stored at 4° C. overnight before reading again after 18hours.

Hit Confirmation by TAMRA Probe in Dose-Response Format.

The primary hits include false positives due to experimental error andfluorescent compounds that fluoresce or absorb in the FTIC emissionrange. After primary screen, a hit confirmation screen was performedusing TAMRA-probe in dose-response format to rid false positives. TAMRAexcites and emits at a longer wavelength range than FITC. The falsepositives caused by overlap of excitation/emission wavelengths of FITCand some fluorescent compounds can be delineated.

Example 3. Screening for Small Molecule Compounds that Bind to Nedd4Proteins

Epic Label-Free Assay Procedure to Measure Binding of Compounds toWWP2/1:

High sensitivity biochemical plates (PerkinElmer Cat. No. 6057468) areused in WWP2 (SEQ ID NO:24) binding assay to maximize proteinimmobilization. The plate was activated before use with 10 μl of 200 mMEDC (N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride)(Sigma Cat. No. 03449)/50 mM sulfo-NHS (N-hydroxysulfosuccinimide)(Pierce Cat. No. 24510) diluted in H₂O. After 30 min incubation at roomtemperature, the plates are washed 3 times with 25 μl H₂O and thencentrifuged inverted at 800 RPM for 1 min. WWP2 (SEQ ID NO:24)immobilization was accomplished by adding 10 μl of 80 μg/ml protein inHEPES buffer (100 mM HEPES, 150 mM NaCl, pH 7.5) followed bycentrifugation at 800 RPM for 1 minute, and then incubated for 3 hoursat room temperature or overnight at 4° C. Plates were then washed threetimes with assay buffer (HEPES buffer with 0.5% DMSO) and then followedwith a final volume of 15 μl assay buffer. The binding assay wasperformed in 4 or 6-point dose-response format. Testing compounds werediluted with assay buffer in 384-well compound plates. Because compoundstocks are in DMSO that affects the Epic label-free readout, thecompound plates were prepared by Echo550 acoustic dispenser to keep DMSOcontent balanced for all wells. Compounds were then transferred to assayplates by a multichannel robotic liquid handler. An alternative approachwas to add compounds in dose-curve format directly to assay plates usingthe nanoliter dispenser Echo550.

The binding of compounds to WWP2 (SEQ ID NO:24) was measured on theEnSpire label-free plate reader. After 25 minutes of thermalequilibration, a baseline reading was taken on the label-free plateswith protein immobilized in assay buffer. In the next step, 25 μl ofreconstituted compounds were added to the plates and mixed. The finalreading was taken after 25 min of thermal equilibration and over aperiod of 60 minutes. The label-free responses were measured as shiftsin reflected wavelength and were expressed in picometers (pm). Resultswere analyzed using the EnSpire label-free user interface software. Thedifference between the last baseline measurements and the signal max wasused to determine the binding of compounds to WWP2.

Example 4: Expression of Soluble TSG101 Fragment Containing PTAP-MotifBinding Domain

The full-length nucleotide and amino acid sequence of TSG101 ispresented in Table VIII. The full-length TSG101 polypeptide (SEQ IDNO:32) is not sufficiently soluble for performing the in vitro screeningassays disclosed herein. Accordingly, a truncated TSG101 peptide thatcontains amino acids corresponding to positions 2-145 of the full-lengthTSG101 peptide and includes a PTAP-binding domain was prepared havingsufficient solubility for these in vitro screening assays. The resultantTSG101 peptide (SEQ ID NO:33) used for these studies is illustrated inTable VIII.

TABLE VIIINucleotide and amino acid sequences of full-length TSG101 peptide andexpression construct for UEV domain of truncated-length TSG101recombinant peptide SEQ ID NO: Sequence¹ 31gaagcggaag tggtgtagtg gtgccgactt cctgttgttt gaggccgggt tgggggtgtgcgattgtgtg ggacggtctg gggcagccca gcagcggctg accctctgcc tgcggggaagggagtcgcca ggcggccgtc ATGgcggtgt cggagagcca gctcaagaaa atggtgtccaagtacaaata cagagaccta actgtacgtg aaactgtcaa tgttattact ctatacaaagatctcaaacc tgttttggat tcatatgttt ttaacgatgg cagttccagg gaactaatgaacctcactgg aacaatccct gtgccttata gaggtaatac atacaatatt ccaatatgcctatggctact ggacacatac ccatataatc cccctatctg ttttgttaag cctactagttcaatgactat taaaacagga aagcatgttg atgcaaatgg gaagatatat cttccttatctacatgaatg gaaacaccca cagtcagact tgttggggct tattcaggtc atgattgtggtatttggaga tgaacctcca gtcttctctc gtcctatttc ggcatcctat ccgccataccaggcaacggg gccaccaaat acttcctaca tgccaggcat gccaggtgga atctctccatacccatccgg ataccctccc aatcccagtg gttacccagg ctgtccttac ccacctggtggtccatatcc tgccacaaca agttctcagt acccttctca gcctcctgtg accactgttggtcccagtag ggatggcaca atcagcgagg acaccatccg agcctctctc atctctgcggtcagtgacaa actgagatgg cggatgaagg aggaaatgga tcgtgcccag gcagagctcaatgccttgaa acgaacagaa gaagacctga aaaagggtca ccagaaactg gaagagatggttacccgttt agatcaagaa gtagccgagg ttgataaaaa catagaactt ttgaaaaagaaggatgaaga actcagttct gctctggaaa aaatggaaaa tcagtctgaa aacaatgatatcgatgaagt tatcattccc acagctccct tatacaaaca gatcctgaat ctgtatgcagaagaaaacgc tattgaagac actatctttt acttgggaga agccttgaga aggggcgtgatagacctgga tgtcttcctg aagcatgtac gtcttctgtc ccgtaaacag ttccagctgagggcactaat gcaaaaagca agaaagactg ccggtctcag tgacctctac TGActtctctgataccagct ggaggttgag ctcttcttaa agtattcttc tcttcctttt atcagtaggtgcccagaata agttattgca gtttatcatt caagtgtaaa atattttgaa tcaataatatattttctgtt ttcttttggt aaagactggc ttttattaat gcactttcta tcctctgtaaactttttgtg ctgaatgttg ggactgctaa ataaaatttg ttgcataaaa aaaaaaaaaa aa 32MAVSESQLKKMVSKYKYRDLTVRETVNVITLYKDLKPVLDSYVFNDGSSRELMNLTGTIPVPYRGNTYNIPICLWLLDTYPYNPPICFVKPTSSMTIKTGKHVDANGKIYLPYLHEWKHPQSDLLGLIQVMIVVFGDEPPVFSRPISASYPPYQATGPPNTSYMPGMPGGISPYPSGYPPNPSGYPGCPYPPGGPYPATTSSQYPSQPPVTTVGPSRDGTISEDTIRASLISAVSDKLRWRMKEEMDRAQAELNALKRTEEDLKKGHQKLEEMVTRLDQEVAEVDKNIELLKKKDEELSSALEKMENQSENNDIDEVIIPTAPLYKQILNLYAEENAIEDTIFYLGEALRRGVIDLDVFLKHVRLLSRKQFQLRALMQKARKTAGLSDLY 33 MGSSHHHHHHSSGLVPRGSHMAS ENLYFQGAVSESQLKKMVSKYKYRDLTVRETVNVITLYKDLKPVLDSYVFNDGSSRELMNLTGTIPVPYRGNTYNIPICLWLLDTYPYNPPICFVKPTSSMTIKTGKHVDANGKIYLPYLHEWKHPQSDLLGLIQVMIVVFGDEPPVFSRP ¹The initiator and terminatorcodons in the full-length TSG101 nucleotide sequence are bolded. The UEVdomain of TSG101 peptide sequence for amino acids 2-145 are presented inbolded fonts in the full-length TSG101 peptide and the truncated-lengthTSG101 recombinant peptide. With respect to the truncated-length TSG101recombinant peptide, the italicized font is leader peptide sequence thatincludes a polyhistidine motif and thrombin cleavage site derived fromthe expression vector, pET28b (Novagen). The underlined sequence is aTEV cleavage site introduced at the amino terminus of the TGS101 peptidesequence.

The complete sequence of the pET28b expression vector that includes thetruncated TSG101 is presented below.

SEQ ID NO: 34:TGGCGAATGGGACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGGGGGCTCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCACCTCGACCCCAAAAAACTTGATTAGGGTGATGGTTCACGTAGTGGGCCATCGCCCTGATAGACGGTTTTTCGCCCTTTGACGTTGGAGTCCACGTTCTTTAATAGTGGACTCTTGTTCCAAACTGGAACAACACTCAACCCTATCTCGGTCTATTCTTTTGATTTATAAGGGATTTTGCCGATTTCGGCCTATTGGTTAAAAAATGAGCTGATTTAACAAAAATTTAACGCGAATTTTAACAAAATATTAACGTTTACAATTTCAGGTGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAATTAATTCTTAGAAAAACTCATCGAGCATCAAATGAAACTGCAATTTATTCATATCAGGATTATCAATACCATATTTTTGAAAAAGCCGTTTCTGTAATGAAGGAGAAAACTCACCGAGGCAGTTCCATAGGATGGCAAGATCCTGGTATCGGTCTGCGATTCCGACTCGTCCAACATCAATACAACCTATTAATTTCCCCTCGTCAAAAATAAGGTTATCAAGTGAGAAATCACCATGAGTGACGACTGAATCCGGTGAGAATGGCAAAAGTTTATGCATTTCTTTCCAGACTTGTTCAACAGGCCAGCCATTACGCTCGTCATCAAAATCACTCGCATCAACCAAACCGTTATTCATTCGTGATTGCGCCTGAGCGAGACGAAATACGCGATCGCTGTTAAAAGGACAATTACAAACAGGAATCGAATGCAACCGGCGCAGGAACACTGCCAGCGCATCAACAATATTTTCACCTGAATCAGGATATTCTTCTAATACCTGGAATGCTGTTTTCCCGGGGATCGCAGTGGTGAGTAACCATGCATCATCAGGAGTACGGATAAAATGCTTGATGGTCGGAAGAGGCATAAATTCCGTCAGCCAGTTTAGTCTGACCATCTCATCTGTAACATCATTGGCAACGCTACCTTTGCCATGTTTCAGAAACAACTCTGGCGCATCGGGCTTCCCATACAATCGATAGATTGTCGCACCTGATTGCCCGACATTATCGCGAGCCCATTTATACCCATATAAATCAGCATCCATGTTGGAATTTAATCGCGGCCTAGAGCAAGACGTTTCCCGTTGAATATGGCTCATAACACCCCTTGTATTACTGTTTATGTAAGCAGACAGTTTTATTGTTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTCCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGAGGAAGCGGAAGAGCGCCTGATGCGGTATTTTCTCCTTACGCATCTGTGCGGTATTTCACACCGCATATATGGTGCACTCTCAGTACAATCTGCTCTGATGCCGCATAGTTAAGCCAGTATACACTCCGCTATCGCTACGTGACTGGGTCATGGCTGCGCCCCGACACCCGCCAACACCCGCTGACGCGCCCTGACGGGCTTGTCTGCTCCCGGCATCCGCTTACAGACAAGCTGTGACCGTCTCCGGGAGCTGCATGTGTCAGAGGTTTTCACCGTCATCACCGAAACGCGCGAGGCAGCTGCGGTAAAGCTCATCAGCGTGGTCGTGAAGCGATTCACAGATGTCTGCCTGTTCATCCGCGTCCAGCTCGTTGAGTTTCTCCAGAAGCGTTAATGTCTGGCTTCTGATAAAGCGGGCCATGTTAAGGGCGGTTTTTTCCTGTTTGGTCACTGATGCCTCCGTGTAAGGGGGATTTCTGTTCATGGGGGTAATGATACCGATGAAACGAGAGAGGATGCTCACGATACGGGTTACTGATGATGAACATGCCCGGTTACTGGAACGTTGTGAGGGTAAACAACTGGCGGTATGGATGCGGCGGGACCAGAGAAAAATCACTCAGGGTCAATGCCAGCGCTTCGTTAATACAGATGTAGGTGTTCCACAGGGTAGCCAGCAGCATCCTGCGATGCAGATCCGGAACATAATGGTGCAGGGCGCTGACTTCCGCGTTTCCAGACTTTACGAAACACGGAAACCGAAGACCATTCATGTTGTTGCTCAGGTCGCAGACGTTTTGCAGCAGCAGTCGCTTCACGTTCGCTCGCGTATCGGTGATTCATTCTGCTAACCAGTAAGGCAACCCCGCCAGCCTAGCCGGGTCCTCAACGACAGGAGCACGATCATGCGCACCCGTGGGGCCGCCATGCCGGCGATAATGGCCTGCTTCTCGCCGAAACGTTTGGTGGCGGGACCAGTGACGAAGGCTTGAGCGAGGGCGTGCAAGATTCCGAATACCGCAAGCGACAGGCCGATCATCGTCGCGCTCCAGCGAAAGCGGTCCTCGCCGAAAATGACCCAGAGCGCTGCCGGCACCTGTCCTACGAGTTGCATGATAAAGAAGACAGTCATAAGTGCGGCGACGATAGTCATGCCCCGCGCCCACCGGAAGGAGCTGACTGGGTTGAAGGCTCTCAAGGGCATCGGTCGAGATCCCGGTGCCTAATGAGTGAGCTAACTTACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCCAGGGTGGTTTTTCTTTTCACCAGTGAGACGGGCAACAGCTGATTGCCCTTCACCGCCTGGCCCTGAGAGAGTTGCAGCAAGCGGTCCACGCTGGTTTGCCCCAGCAGGCGAAAATCCTGTTTGATGGTGGTTAACGGCGGGATATAACATGAGCTGTCTTCGGTATCGTCGTATCCCACTACCGAGATATCCGCACCAACGCGCAGCCCGGACTCGGTAATGGCGCGCATTGCGCCCAGCGCCATCTGATCGTTGGCAACCAGCATCGCAGTGGGAACGATGCCCTCATTCAGCATTTGCATGGTTTGTTGAAAACCGGACATGGCACTCCAGTCGCCTTCCCGTTCCGCTATCGGCTGAATTTGATTGCGAGTGAGATATTTATGCCAGCCAGCCAGACGCAGACGCGCCGAGACAGAACTTAATGGGCCCGCTAACAGCGCGATTTGCTGGTGACCCAATGCGACCAGATGCTCCACGCCCAGTCGCGTACCGTCTTCATGGGAGAAAATAATACTGTTGATGGGTGTCTGGTCAGAGACATCAAGAAATAACGCCGGAACATTAGTGCAGGCAGCTTCCACAGCAATGGCATCCTGGTCATCCAGCGGATAGTTAATGATCAGCCCACTGACGCGTTGCGCGAGAAGATTGTGCACCGCCGCTTTACAGGCTTCGACGCCGCTTCGTTCTACCATCGACACCACCACGCTGGCACCCAGTTGATCGGCGCGAGATTTAATCGCCGCGACAATTTGCGACGGCGCGTGCAGGGCCAGACTGGAGGTGGCAACGCCAATCAGCAACGACTGTTTGCCCGCCAGTTGTTGTGCCACGCGGTTGGGAATGTAATTCAGCTCCGCCATCGCCGCTTCCACTTTTTCCCGCGTTTTCGCAGAAACGTGGCTGGCCTGGTTCACCACGCGGGAAACGGTCTGATAAGAGACACCGGCATACTCTGCGACATCGTATAACGTTACTGGTTTCACATTCACCACCCTGAATTGACTCTCTTCCGGGCGCTATCATGCCATACCGCGAAAGGTTTTGCGCCATTCGATGGTGTCCGGGATCTCGACGCTCTCCCTTATGCGACTCCTGCATTAGGAAGCAGCCCAGTAGTAGGTTGAGGCCGTTGAGCACCGCCGCCGCAAGGAATGGTGCATGCAAGGAGATGGCGCCCAACAGTCCCCCGGCCACGGGGCCTGCCACCATACCCACGCCGAAACAAGCGCTCATGAGCCCGAAGTGGCGAGCCCGATCTTCCCCATCGGTGATGTCGGCGATATAGGCGCCAGCAACCGCACCTGTGGCGCCGGTGATGCCGGCCACGATGCGTCCGGCGTAGAGGATCGAGATCTCGATCCCGCGAAATTAATACGACTCACTATAGGGGAATTGTGAGCGGATAACAATTCCCCTCTAGAAATAATTTTGTTTAACTTTAAGAAGGAGATATACC

GGCAGCAGCCATCATCATCATCATCACAGCAGCGGCCTGGTGCCGCGCGGCAGCCATATGGCTAGCgaaaacctgtacttccagggcgcggtgtcggagagccagctcaagaaaatggtgtccaagtacaaatacagagacctaactgtacgtgaaactgtcaatgttattactctatacaaagatctcaaacctgttttggattcatatgtttttaacgatggcagttccagggaactaatgaacctcactggaacaatccctgtgccttatagaggtaatacatacaatattccaatatgcctatggctactggacacatacccatataatccccctatctgttttgttaagcctactagttcaatgactattaaaacaggaaagcatgttgatgcaaatgggaagatatatcttccttatctacatgaatggaaacacccacagtcagacttgttggggcttattcaggtcatgattgtggtatttggagatgaacctccagtcttctctcgtccttgataaGGATCCGAATTCGAGCTCCGTCGACAAGCTTGCGGCCGCACTCGAGCACCACCACCACCACCACTGAGATCCGGCTGCTAACAAAGCCCGAAAGGAAGCTGAGTTGGCTGCTGCCACCGCTGAGCAATAACTAGCATAACCCCTTGGGGCCTCTAAACGGGTCTTGAGGGGTTTTTTGCTGAAAGGAGGAACTATATCCGGATwherein the bold font denote the locations of the initiator andterminator codons for the recombinant peptide pro-form; the upper caseletters denote pET28b vector sequences; the lower case letter denote thetruncated TSG101 (2-145) coding sequences, the italicized font includesthe leader peptide coding sequence that includes a polyhistidine motifand thrombin cleavage site derived from the expression vector, pET28b(Novagen). The underlined sequence is a TEV cleavage site introduced atthe amino terminus of the TGS101 peptide sequence.

Example 5: High Throughput Fluorescence-Based Thermal Shift (FTS) Assayfor TSG101

The NU-HTA has developed a robotic pipeline for small molecule proteinligand screening by FTS. See Luan C H, Light S H, Dunne S F, Anderson WF. Ligand screening using Fluorescence Thermal Shift Analysis (FTS). InStructural Genomics and Drug Discovery: Methods and Protocols. MethodsMol. Biol. 2013, Humana Press (In press), which is incorporated byreference in its entirety. The pipeline uses an Echo550 acoustictransfer robot (Labcyte, CA) for compound addition and the Mosquitorobot (TTP LabTechnologies, UK) for protein dispensing followed bythermal scanning coupled with fluorescence detection which is performedon a real-time PCR machine CFX384 (Bio-Rad Laboratories). The methoddoes not require any labeling on either protein or compound. Afluorescent dye Sypro-Orange (Invitrogen) is used for assay detection.TSG101 (SEQ ID NO:33) has a thermal unfolding profile ideal for usingFTS as primary screen assay.

Compound Library Screening.

For primary screening, the TSG101 protein (SEQ ID NO:33) was premixed ata concentration of 2 uM with a 5× concentration of Sypro-Orange in Hepesbuffer (100 mM HEPES, 150 mM NaCl, pH 7.5). Then 10 uL of theprotein-dye mix was added to an assay plate. And 10 to 50 nanoliters ofcompound, equal to 10 to 50 uM, were added. The plate was shaken toensure proper mixing and then sealed with optical seal and centrifuged.The thermal scan was performed from 10 to 95° C. with a temperature ramprate of 1.5° C./min. The fluorescence was recorded every 10 sec. Dataanalysis and report generation were performed by using the in-housesoftware excelFTS. Hit compounds identified were tested in dose-responseformat.

Validation of the Confirmed Hits with TSG101 by FP and EpicLF.

The confirmed hits from the primary FTS screening were validated byfluorescent polarization (FP) and Epic label-free (EpicLF) assays. TheEpicLF assay was performed on an EnSpire multifunction plate reader(Perkin Elmer) to determine the Kd of hit compound binding to TSG101(SEQ ID NO:33) as described generally in Example 3 for candidatecompound binding to Nedd 4 peptide family members. The hit compoundswere also tested by FP assay to determine the IC50 of disrupting TSG101(SEQ ID NO: 33) and PTAP-probe interaction as described generally inExample 2 for candidate compound screening to determine IC50 ofdisrupting Nedd 4 peptide family members and PY-probe interaction.

For this purpose, tandem-linked versions of the PTAP motif from HIV-1gag was designed and synthesized for use in these experiments. Thestructures of the resulting peptides are presented below.

SEQ ID NO: 20: RPGNFLQSRPEPTAPPFLQSRPEPTAPPEESFRRRR SEQ ID NO: 21:FITC-RPGNFLQSRPEPTAPPFLQSRPEPTAPPEESFRRRR SEQ ID NO: 22:TAMRA-RPGNFLQSRPEPTAPPFLQSRPEPTAPPEESFRRRR

Example 6: In Vivo Screening for Compounds that Disrupt the Interactionof TSG101 and the PTAP L-Domain Sequence in the p6 Region of HIV-1 Gag(Prophetic Example)

Cells will be transfected with suitable vector constructs to express(either transiently or stably) EGFP reconstituted from a two-hybridsystem comprising a fusion protein containing PTAPP motifs and theN-terminal portion of EGFP and a fusion protein containing TSG101polypeptides and the C-terminal portion of EGFP. Following establishmentof stable EGFP expression, the ability of test compound to enter thecells and inhibit EGFP expression will be evaluated. A reduction of EGFPfluorescence as a function of test compound administration to cells willindicate that the compound inhibits formation of functional GFPcomplexes from the two component system. Controls will be performed thatinclude evaluation the cytotoxicity of the test compounds havingpositive effect in this assay.

Plasmids for CEGFP-N1.

The plasmid pCEGFP-N1, which places CEGFP under control of the T7promoter, was created by PCR amplification (Deep Vent polymerase) of thegene for C-terminal EGFP (from 159 to 265 amino acids) from pEGFP-N1using the oligonucleotides 5′-ataggatccaccgg tcgccaccggtggctctggcaagaacgg catcaaggtg aacttcaa-3′ forward primer (SEQ ID NO:35) and5′-gtcgcggccgctttacttgtacagctcgtccatg-3′ reverse primer (SEQ ID NO:36).The 4-residue linker (GGSG (SEQ ID NO:37)) is located at the beginningof the CEGFP gene. This was followed by digestion of PCR products andthe plasmid pEGFP-N1 with NheI and XhoI, and then ligation with T4 DNAligase. The C-terminal EGFP ligation was confirmed by DNA sequencing byusing sequencing primer (5′-gcagagctggtttagtg-3′ forward (SEQ ID NO:38),from 561 to 577 bp of pEGFP-N1 sequences).

Plasmids for NEGFP-C3.

The plasmid pNEGFP-N1, which places NEGFP under control of the T7promoter, was created by PCR amplification (Deep Vent polymerase) of thegene for N-terminal EGFP (from 1 to 158 amino acids) from pEGFP-C3 usingthe oligonucleotides 5′-cagatcc gctagcgctaccggtcgcca ccatggtgag forwardprimer (SEQ ID NO:39) and5′-atactcgagatctgagtacccagagccagagccaccctgatgtcggccatgatatag-3′ reverseprimer (SEQ ID NO:40). The 6-residue peptide linker (GGSGSG [(SEQ IDNO:41)]) is located at the end of the NEGFP gene. This was followed bydigestion of PCR products and the plasmid pEGFP-C3 with BamHI and NotI,and then ligation with T4 DNA ligase. The N-terminal EGFP ligation wasconfirmed by DNA sequencing by using sequencing primer5′gtgggaggttttttaaa-3′ reverse (from 1451 to 1467 bp of pEGFP-C3sequences) (SEQ ID NO:42).

Construct NEGFP-2×PTAPP.

Two copies of PTAPP sequences of HIV Gag were amplified from pGBT9_HIVGag with 2PTAP template (from Dr. Carol Carter's lab) by PCR reaction byusing the oligonucleotides. This was followed by digestion of PCRproducts and the plasmid pNEGFP-C3 with EcoRI and BamHI, and thenligation with T4 DNA ligase.

Construct CEGFP-TSG101.

TSG101 was amplified by PCR reaction by using the oligonucleotides. Thiswas followed by digestion of PCR products and the plasmid pNEGFP-C3 withEcoRI and BamHI, and then ligation with T4 DNA ligase.

Example 7: Biological Testing of Compounds to Inhibit Virus BuddingDetected by Release of VLPs from Cells

Transfection of 293/E Cells and Chemical Inhibitor Treatment.

293/E cells were cultured in DMEM supplemented with 10% fetal bovineserum, penicillin (1,000 units/ml), and streptomycin (1,000 μg/ml) to50% confluence at 37° C. Expression of plasmids from p2036 was high in293/E cells because these cells stably express the EBNA1 protein of EBVand the p2036 constructs contain the EBV FR plasmid maintenance elementthat EBNA1 binds. Therefore 293/E cells were used when proteinsexpressed from p2036 were to be detected by western analysis. In allexperiments, 24-well plates of 293/E cells were transfected with 0.5 μgof p2036-ASLV Gag with the X-treme Gene9 transfection reagent (RocheDiagnostics, Alameda, Calif., USA) according to the manufacturer'sinstructions. After 24 h after DNA transfection, the cells were washedwith 1×DPS that contained CaCl₂ (0.1 mM) and MgCl₂ (1 mM) [Gibco#14040-133], and 1 ml of cell culture medium (10% FBS, 1%Penicillin/Streptomycin) containing CaCl₂ (0.5 mM) and MgCl₂ (5 mM) wasadded. Thereafter, the inhibitor compounds were added to the culturemedium of parallel culture wells at one of the following finalconcentrations: 5 μM, 10 μM, 20 μM, or 40 μM and the cells remained incontact with the culture medium containing the inhibitor compounds for 5hr. Cells and virus-like particles (VLPs) released into the cell mediawere collected 5 h after chemical treatment.

Detection of Gag Proteins by Western Blotting.

For the budding assay, both media and cell lysate fractions werecollected. The cell lysate fractions were prepared by suspension inradioimmune precipitation assay (RIPA) buffer [PBS containing 1% NonidetP-40, 0.5% sodium deoxycholate, 0.1% SDS, and protease inhibitor mixturetablets] 48 hours post-transfection. VLPs were purified from the cellculture medium by centrifugation through a 20% sucrose cushion at100,000×g for 1 h at 4° C. (Beckman SW50.1 rotor). The pelleted VLPswere suspended in 100 μl of RIPA buffer containing protease inhibitormixture tablets. For lysate fractions, Gag proteins wereimmunoprecipitated overnight at 4° C. with an anti-ASLV monoclonal serum(1:500-1:1000 dilution) and 20 μl of protein A-agarose beads. Theprecipitated proteins were separated by SDS-PAGE and transferred to apolyvinylidene difluoride membrane. After blocking of the membrane withwash buffer (10 mM Tris-HCl, pH 8.0, 150 mM NaCl, and 0.1% Tween 20)containing 5% nonfat dry milk, ASLV Gag proteins were detected with anAMV MA (p19) directed monoclonal antibody (mAb) and an anti-mouseIgG-HRP secondary antibody for ECL (Denville Scientific; Metuchen,N.J.).

Viral Replication Assays.

293/E cells in 6-well plates were transfected with HIV-1 luciferasereporter vector consisting of the following plasmids: 1.5 μg of pHIVenv-Luc and 0.75 μg of VSV-G (Provided by Tom Hope, NorthwesternUniversity). Supernatants containing pseudo-typed virions were harvested24 or 48 h after the chemical treatments, and the cells were harvestedat the same time for western blot analyses. To measure titers, virusparticles were filtered through a 0.45 μm filter, and infectivity wasassayed by incubating in duplicate 1×10⁵ 293/E cells/well with 1 ml ofpseudo typed virus in 6-well plates for 5 h. Virus was then removed andcell growth media was added. The cells were lysed in 400 μl of cellculture lysis reagent (Promega, Madison, Wis.) at 72 hpost-transfection. The luciferase activity was measured with aluciferase assay kit (Promega, Madison, Wis.) and a FB12 luminometeraccording to manufacturer's instructions. The data are presented as theaverage of the duplicates.

Example 8: Biological Testing of Nedd4 Inhibitor Candidate Compounds K21(Benserazide Hydrochloride) and N20 (Oxytetracycline) by Cell CultureAssays In Vitro

Cell-Based Inhibition Assay

COS-1 cells were seeded on twelve-well plates and grown in 1 ml of DMEMwith 10% fbs and 1% Penicillin/Streptomycin. The following day, wellwere confluency was ˜60% were selected for co-transfection with an NL4-3derived construct (pdeltaEnv) and pIIIEnv for expression of HIV-1virus-encoded proteins. Inhibitor treatment was done on two sets oftransfected cells: one, at 5 hr post-transfection and the other, at 24hours post-transfection. In each case, the tissue culture media wasremoved and replaced with treatment Nedd4 inhibitor (100 μM finalconcentration for K21 and 20 μM final concentration for N20) or DMSOcontrol (1% final concentration) in DMEM with 10% FBS, 1%Penicillin/Streptomycin, 5 mM MgCl₂, and 0.5 mM CaCl₂. After a 24-hrtreatment period, the tissue culture media was collected, cleared ofdebris by running through a 0.45 um syringe filter and analyzed foramount of virus particles by Elisa p24 capture assay.

Cell Death Evaluation.

The standard trypan blue assay, where dead cells are blue whenvisualized under a light microscope, was used to determine the percentof dead cells in the culture. Briefly, cells from a well ofDMSO-treated, K21-treated and N20-treated samples were dislodged fromthe well with a stream of 1 ml PBS, collected, pelleted at 200 rpm for 2min, and suspended in 300 μl trypsin-EDTA, incubated at 37° C. for 30min. A diluted cell suspension was prepared by mixing 200 μl of thetrypsin-treated cell suspension and 800 μl of PBS was used for theassay. For the assay itself: from the 1 ml of diluted cell suspension,50 μl was taken an placed on paraffin and to this was mixed in 50 μl of0.4% Trypan Blue dye in PBS, after exactly 2 minutes later, the mixturewas loaded on a hematocytometer. The hematocytometer was placed on thelight microscope stage and a blue cell count and an all cells count(blue and not blue cells) obtained. Two 50 μl aliquots were counted persample. The results are presented in Table VII.

TABLE VII Cytotoxicity results for contacting cells with inhibitorcompounds. Compound Blue cells Total cells Cytotoxicity¹ DMSO 7 29 24% 949 18% (21%) K21 13 38 34% 17 60 28% (31%) N20 11 37 29% 7 20 35% (32%)¹Cytotoxicity (%) is determined by the fraction (blue cells)/(totalcells) multiplied by 100 (%). The average of the independent experimentsis shown in parentheses.

Cell Growth Based on Cell Lysate Actin Levels

The levels of actin of cell lysates prepared from DMSO-treated,K21-treated and N20-treated cells were determined by SDS-PAGE followedby Western analysis where the immunoblot were probed with mouseanti-actin antibody. The intensity of the actin bands were essentiallycomparable for all samples (not shown).

Effects on HIV-1 Production Accumulation of Gag-Related Proteins in theCell

The levels of Gag-related proteins in cell lysates wereimmune-precipitated with polyclonal anti CA antibody. Proteins in theimmune-precipitate were separated by SDS-PAGE and followed by Westernanalysis where the blot was probed with mouse anti-CA antibody. Thecommercial antibody used (NEN NEA-9306) is known to recognize the Gagprecursor better than the mature p24 proteins (Dietrich et al 2001).Both GagPr55 and mature p24, as adjudged by their respective bandintensities, were essentially comparable for all samples (not shown).

Elisa p24 Capture Assay of Tissue Culture Media

At 100 μM, K21 was inhibitory to virus particle release. There was areduction in the amount of virus particle detected in the tissue culturemedia. This was true whether the inhibitor was added at 5 hrspost-transfection or at 24 hrs post transfection. At 20 μM, N20 was alsoexerted an inhibitory effect on virus particle release but it wasdifferent from that of K21. There was a reduction in the amount of virusparticle detected in the tissue culture media when inhibitor was addedat 5 hr post-transfection. This reduction was not seen when inhibitorwas added 24 hrs post-transfection.

Specific Infectivity

The amount of infectious virus normalized to ng of p24 obtained from theELISA assay in the tissue cultures was determined by multinuclearactivation of a galactosidase indicator (MAGI) assay. In this assay,infectious unit is scored by the blue color that is assumed by infectedMAGI cells (Hela cells that have been engineered to express theindicator when the infecting particle is able to simulated naturalinfection up until expression of HIV-1 Tat). Counts of Blue cells per ngp24 for the DMSO controls, K21-treated and N20-treated samples werecomparable.

Example 9: HSV-1 Inhibition Assay by PTAP-Inhibitor Compounds F15(Esomeprazole Potassium) and N16 (Tenatoprazole) MTS Assay

The cytotoxicity of the compounds was determined by a commerciallyavailable assay (Celltiter 96® AQ_(ueous) One Solution cellproliferation assay reagent; Promega, Madison, Wis.), as describedpreviously (Akkarawongsa et al., 2006). The CellTiter 96® AQ_(ueous) OneSolution Cell Proliferation Assay is a colorimetric method fordetermining the number of viable cells in proliferation, cytotoxicity orchemosensitivity assays. The CellTiter 96® AQ_(ueous) One SolutionReagent contains a tetrazolium compound[3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium,inner salt; MTS] and an electron coupling reagent (phenazineethosulfate; PES). PES has enhanced chemical stability, which allows itto be combined with MTS to form a stable solution. The CellTiter 96®AQ_(ueous) Assay uses phenazine methosulfate (PMS) as the electroncoupling reagent, and PMS Solution and MTS Solution are suppliedseparately. PES has enhanced chemical stability, which allows it to becombined with MTS to form a stable solution. Assays are performed byadding a small amount of the CellTiter 96® AQ_(ueous) One SolutionReagent directly to culture wells, incubating for 1-4 hours and thenrecording absorbance at 490 nm with a 96-well plate reader. The quantityof formazan product as measured by the amount of 490 nm absorbance isdirectly proportional to the number of living cells in culture.

Briefly, Vero cells (1.5×10⁴ cells/well) were seeded in a 96-well plate,and the plate was incubated for 24 h at 37° C. A total of 20 μl ofmedium containing the desired concentration of inhibitors was added tothe cells. Control cells received medium only. After incubation of thecells in the presence of peptide overnight at 37° C., 20 μl of the 96AQqueous One Solution cell proliferation assay reagent was added to eachwell. The plate was then incubated for 2 h at 37° C., and the absorbanceat 490 nm was determined with a 96-well plate reader (Perkin Elmer).

Assay for Inhibitor Compound Effects on Infected Cells and HSV-1 VirusProduction and Release.

Vero cells were grown in DMEM supplemented with 10% FBS. Viralinfections were performed in DMEM supplemented with 1% heat-inactivatedFBS. Vero cells were inoculated HSV-1 (strain F) at indicatedconcentrations, after 2 h, the cells were washed and treated with 0.1 Msodium citrate buffer (pH 3.0) for 1 min to inactivate unpenetratedviruses, washed again and incubated with fresh medium containing 1%heat-inactivated FBS. At different times after inoculation, one-half ofthe medium was harvested as a supernatant sample and the cells werescraped into the rest of the half medium containing released viruses asa total virus sample and lysed by sonication. Then virus titers weredetermined by standard plaque assay on Vero cells.

As shown in FIG. 8, inhibitor compound F15 (Esomepazole) resulted ininfectious HSV-1 virion release from HSV-1 infected cells at about 10%of the level observed for virus release from infected cells notcontacted with an inhibitor compound at the higher concentration tested(that is, more than a 90% reduction in virus release when F15 is presentat 80 μM in the culture medium). Inhibitor compound F15 also reduced thetotal load of infectious virions produced in the cells, whether releasedor not from the cells, to about 25% of the level observed for virusrelease from infected cells not contacted with an inhibitor compound atthe higher concentration tested (that is, more than a 75% reduction intotal infectious virus when F15 is present at 80 μM in the culturemedium). As shown in FIG. 8A, inhibitor compound N16 (Tenatoprazole)reduced infectious HSV-1 virion release from HSV-1 infected cells atabout 5% of the level observed for virus release from infected cells notcontacted with an inhibitor compound at the higher concentration tested(that is, more than a 95% reduction in virus release when N16 is presentat 80 μM in the culture medium). Thus, inhibitor compound N16 wasslightly more effective than F15 at reducing infectious HSV-1 virionparticle release from HSV-1 infected cells at the concentrations tested.Neither inhibitor compound was cytotoxic to the Vero cells at theconcentrations tested (FIG. 8B).

Example 10: Testing of Compounds to Inhibit the Budding of KSHV(Prophetic Example)

KSHV, a member of the herpes virus family, buds from cells with a Vps4dependence, probably using the PY motif-dependent pathway. To test theeffect of inhibitors on KSHV release, of a recently reported cell line(iSLK.219) will be used that allows the doxycycline (Dox) inducibleexpression of the KSHV lytic transactivator protein (RTA) and isinfected with recombinant KSHV.219. See Myoung J, Ganem D. Generation ofa doxycycline-inducible KSHV producer cell line of endothelial origin:maintenance of tight latency with efficient reactivation upon induction.J Virol Methods. 2011, 174(1-2):12-21. PMCID: 3095772 and Vieira J,O'Hearn P M. Use of the red fluorescent protein as a marker of Kaposi'ssarcoma-associated herpesvirus lytic gene expression. Virology. 2004;325(2):225-40. The contents of these printed publication areincorporated by reference in their entirety. Upon Dox treatment of thesecells, lytic reactivation results in the production of infectious KSHVcarrying a GFP reporter cassette. The ability of individual dominantnegative ESCRT proteins interfere with the release of infectious KSHVwill be evaluated initially. For this purpose, 10⁵ iSLK.219 cells willbe plated per well in 6 well plates and transfected the next day withincreasing amounts of control vectors or vectors expressing dominantnegative ESCRT proteins (range 0.2 μg-2 mg/well) using Lipofectamine2000 (Invitrogen) as instructed. Four hours after transfection, growthmedium will be exchanged for medium containing 1 mg/ml Dox and cellswill be incubated for 48 hours. Resulting virus containing supernatantwill be cleared by centrifugation (5 min, 2000 rpm), filtered through450 nm pore size filters and will titered on the KSHV-negativeendothelial cell line SLK by serial dilutions in a 24 well format. Forthis, 20,000 cells will be infected with filtered virus in serialdilutions into normal growth medium (i.e. 1:1, 1:5, 1:25). Forty-eighthours after infection, SLK cells will be trypsinized, recovered bycentrifugation (5 min, 1400 rpm), fixed with 4% paraformaldehyde for 20min at room temperature, washed once with PBS, suspended in 300 ml PBSand subjected to flow cytometry on a FACS Canto II in order to establishthe percentage of green cells. If ESCRT proteins are indeed required forKSHV production, a reduction in virus titer will be expected.

In a parallel positive control experiment, the effect of wild type ordominant negative ESCRT proteins on the release of a GFP-positivelentiviral vector pLCE from SLK cells will be monitored by titration andflow cytometry. The lentiviral vector pLCE is described in Zhang J, JimaD D, Jacobs C, Fischer R, Gottwein E, Huang G, et al. Patterns ofmicroRNA expression characterize stages of human B-cell differentiation.Blood. 2009; 113(19):4586-94, the contents of which are incorporated byreference in its entirety. This experiment will be conducted as outlinedabove, except that ESCRT vectors will be co-transfected with 0.5 mg pLCEand 0.125 mg each of three packaging plasmids (pMDLgpRRE, pRSV-Rev andpVSV-G). Because lentiviral release depends on the ESCRT machinery, weexpect that dominant negative ESCRT vectors will result in a reductionof lentiviral titers. If a dependence of KSHV budding on the ESCRTmachinery can be established in the experiments outlined above, iSLK.219cells will be used to test the effectiveness of novel small moleculeinhibitors using the experimental procedure described above.

DEFINITIONS

When introducing elements of aspects of the embodiments, the articles“a,” “an,” “the,” and “said” are intended to mean that there are one ormore of the elements. The terms “comprising,” “including,” and “having”are intended to be inclusive and mean that there may be additionalelements other than the listed elements. The word “or” means any onemember of a particular list and also includes any combination of membersof that list, unless otherwise specified.

The modal verb “may” refers to the preferred use or selection of one ormore options or choices among several described embodiments or featurescontained within the same. Where no options or choices are disclosedregarding a particular embodiment or feature contained in the same, themodal verb ‘“may” refers to an affirmative act regarding how to make oruse an aspect of a described embodiment or feature contained in thesame, or a definitive decision to use a specific skill regarding adescribed embodiment or feature contained in the same. In this lattercontext, the model verb “may” has the same meaning and connotation asthe auxiliary verb “can.”

The term “about” is used herein to mean approximately, roughly, around,or in the region of. When the term “about” is used in conjunction with anumerical range, it modifies that range by extending the boundariesabove and below the numerical values set forth. Preferably, the term“about” is used herein to modify a numerical value above and below thestated value by a variance of 20 percent up or down (higher or lower).

The term “associative complex” refers to two or more molecular entities(for example, two separate polypeptides; an isolated compound and anisolated polypeptide; two separate fusion proteins that interact in atwo-hybrid system, two separate proteins, among others) that are presentin a complex or that are capable of forming a complex.

The chemical structures described herein are named according to IUPACnomenclature rules and include art-accepted common names andabbreviations where appropriate. The IUPAC nomenclature can be derivedwith chemical structure drawing software programs, such as ChemDraw®(PerkinElmer, Inc.), ChemDoodle® (iChemLabs, LLC) and Marvin (ChemAxonLtd.). The chemical structure controls in the disclosure to the extentthat an IUPAC name is misnamed or otherwise conflicts with the chemicalstructure disclosed herein.

The chemical structures described herein are also cataloged according toCAS Registry Nos. where appropriate. The chemical structure controls inthe disclosure to the extent that an CAS Registry No. is misidentifiedor otherwise conflicts with the chemical structure disclosed herein.

In view of the above, it will be seen that several advantages of theinvention are achieved and other advantageous results attained.

Not all of the depicted components illustrated or described may berequired. In addition, some implementations and embodiments may includeadditional components. Variations in the arrangement and type of thecomponents may be made without departing from the spirit or scope of theclaims as set forth herein. Additional, different or fewer componentsmay be provided and components may be combined. Alternatively or inaddition, a component may be implemented by several components.

The above description illustrates the invention by way of example andnot by way of limitation. This description clearly enables one skilledin the art to make and use the invention, and describes severalembodiments, adaptations, variations, alternatives and uses of theinvention, including what is presently believed to be the best mode ofcarrying out the invention. Additionally, it is to be understood thatthe invention is not limited in its application to the details ofconstruction and the arrangement of components set forth in thefollowing description or illustrated in the drawings. The invention iscapable of other embodiments and of being practiced or carried out invarious ways. Also, it will be understood that the phraseology andterminology used herein is for the purpose of description and should notbe regarded as limiting.

Having described aspects of the invention in detail, it will be apparentthat modifications and variations are possible without departing fromthe scope of aspects of the invention as defined in the appended claims.As various changes could be made in the above constructions, products,and methods without departing from the scope of aspects of theinvention, it is intended that all matter contained in the abovedescription and shown in the accompanying drawings shall be interpretedas illustrative and not in a limiting sense.

All references, citations, patent applications, patent publicationsspecifically mentioned in this disclosure are hereby incorporated byreference in their entireties.

1-10. (canceled)
 11. A method of inhibiting release of an envelopedvirus from a cell, comprising contacting the cell with a compound havingan antiviral activity, said antiviral activity comprises: (i) inhibitingformation of an associative complex; or (ii) disrupting formation of anassociative complex, wherein the associative complex comprises anL-domain motif of the enveloped virus and at least one cellularpolypeptide, or fragment thereof, capable of binding the L-domain motifof the enveloped virus.
 12. The method of claim 11, wherein L-domainmotif comprises at least one of a PY-motif or a PTAP-motif.
 13. Themethod of claim 11, wherein L-domain motif comprises at least one memberselected from the group consisting of SEQ ID NOS: 1-22.
 14. The methodof claim 11, wherein the at least one cellular polypeptide comprises anESCRT complex protein.
 15. The method of claim 14, wherein the ESCRTcomponent protein comprises at least one member selected from a Nedd4-related family peptide and TSG101, fragments thereof, and combinationsthereof.
 16. The method of claim 14, wherein ESCRT component proteincomprises one of SEQ ID NOS: 24, 29, 32 and
 33. 17. The method of claim11, wherein the enveloped virus comprises at least one member selectedfrom the group consisting of Lassa fever virus, lymphocyticchoriomeningitis virus, Ebola virus, Marberg virus, hepatitis B virus,Herpes simplex virus, type 1, Herpes simplex virus, type 2,cytomegalovirus, Simian virus, type 5, Mumps virus, avian sarcomaleucosis virus, human immunodeficiency virus, type 1, humanT-lymphotrophic virus, type 1, equine infectious anemia virus, vesicularstomatitis virus, rabies virus and combinations thereof.
 18. The methodof claim 11, wherein the cell comprises a host cell for supportingactive replication of the enveloped virus.
 19. The method of claim 11,wherein the compound comprises at least one member selected from thegroup consisting of compounds having the structure of formulas(VII)-(XIII):

and combinations thereof.
 20. (canceled)